U.S. patent number 9,512,192 [Application Number 15/078,885] was granted by the patent office on 2016-12-06 for collagen-binding synthetic peptidoglycans, preparation, and methods of use.
This patent grant is currently assigned to Purdue Research Foundation. The grantee listed for this patent is Purdue Research Foundation. Invention is credited to Steve Higbee, John Eric Paderi, Alyssa Panitch, Kinam Park, Katherine Allison Stuart.
United States Patent |
9,512,192 |
Panitch , et al. |
December 6, 2016 |
**Please see images for:
( Certificate of Correction ) ** |
Collagen-binding synthetic peptidoglycans, preparation, and methods
of use
Abstract
This invention relates to collagen-binding synthetic
peptidoglycans and engineered collagen matrices comprising a
collagen matrix and a collagen-binding synthetic peptidoglycan
where the collagen-binding synthetic peptidoglycan can be aberrant
or can have amino acid homology with a portion of the amino acid
sequence of a protein or a proteoglycan that regulates collagen
fibrillogenesis. The invention also relates to kits, compounds,
compositions, and engineered graft constructs comprising such
collagen-binding synthetic peptidoglycans or engineered collagen
matrices and methods for their preparation and use.
Inventors: |
Panitch; Alyssa (Davis, CA),
Paderi; John Eric (San Francisco, CA), Park; Kinam (West
Lafayette, IN), Stuart; Katherine Allison (West Lafayette,
IN), Higbee; Steve (West Lafayette, IN) |
Applicant: |
Name |
City |
State |
Country |
Type |
Purdue Research Foundation |
West Lafayette |
IN |
US |
|
|
Assignee: |
Purdue Research Foundation
(West Lafayette, IN)
|
Family
ID: |
41114790 |
Appl.
No.: |
15/078,885 |
Filed: |
March 23, 2016 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20160244495 A1 |
Aug 25, 2016 |
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Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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14466889 |
Aug 22, 2014 |
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12934551 |
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8846003 |
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PCT/US2009/038624 |
Mar 27, 2009 |
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61081984 |
Jul 18, 2008 |
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61039933 |
Mar 27, 2008 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61L
27/54 (20130101); C07K 14/4725 (20130101); C07K
14/78 (20130101); A61P 43/00 (20180101); C07K
14/00 (20130101); A61P 7/00 (20180101); A61L
27/227 (20130101); A61L 27/3804 (20130101); A61P
7/02 (20180101); C07K 9/00 (20130101); C07K
9/001 (20130101); A61L 27/24 (20130101); A61K
38/00 (20130101); A61L 2300/42 (20130101); A61L
2300/25 (20130101) |
Current International
Class: |
C07K
9/00 (20060101); C07K 14/78 (20060101); A61K
31/726 (20060101); C07K 14/47 (20060101); A61L
27/24 (20060101); A61L 27/54 (20060101); C07K
14/00 (20060101); A61L 27/22 (20060101); A61K
38/14 (20060101); A61L 27/38 (20060101); A61K
38/00 (20060101) |
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|
Primary Examiner: Desai; Anand
Assistant Examiner: Liu; Samuel
Attorney, Agent or Firm: Sheppard Mullin Richter &
Hampton LLP
Government Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
Part of the work during the development of this invention was made
with government support from the National Institutes of Health
under grant application number K25HL074968. The U.S. Government has
certain rights in the invention.
Parent Case Text
CROSS REFERENCE TO RELATED APPLICATIONS
This application is a continuation of U.S. application Ser. No.
14/466,889, filed Aug. 22, 2014, which is a continuation of U.S.
application Ser. No. 12/934,551, filed Sep. 24, 2010, now U.S. Pat.
No. 8,846,003, which is a U.S. national stage application filed
under 37 C.F.R. .sctn.371 of International Application No.
PCT/US2009/038624, filed Mar. 27, 2009, which claims priority to
U.S. Provisional Patent Application No. 61/039,933 filed Mar. 27,
2008, and U.S. Provisional Patent Application No. 61/081,984 filed
Jul. 18, 2008, the entire disclosures of which are hereby
incorporated by reference.
Claims
What is claimed is:
1. A synthetic peptidoglycan comprising a glycan and from 1 to 50
collagen binding peptides, each of which is covalently bonded to
the glycan, wherein the peptides comprise amino acids 1-18
(RRANAALKAGELYKSILY) of SEQ ID NO: 1.
2. The peptidoglycan of claim 1, wherein the peptides comprise SEQ
ID NO: 1.
3. The peptidoglycan of claim 1, wherein the peptides are bonded to
the glycan via a linker having a molecular weight of about 20 to
about 500 Daltons.
4. The peptidoglycan of claim 1, wherein the glycan is a
glycosaminoglycan.
5. The peptidoglycan of claim 1, wherein the glycan is alginate,
agarose, chondroitin, dermatan, dermatan sulfate, heparan, heparin,
keratin, or hyaluronan.
6. The peptidoglycan of claim 1, wherein the glycan is dermatan
sulfate.
7. The peptidoglycan of claim 1, wherein the glycan is heparin.
8. The peptidoglycan of claim 1, wherein the peptidoglycan
comprises from 1 to 10 of the peptides.
9. A composition comprising the peptidoglycan of claim 1 and a
pharmaceutically acceptable excipient or diluent, or a combination
thereof.
10. A synthetic peptidoglycan comprising dermatan sulfate and from
1 to 10 collagen binding peptides, all of which are covalently
bonded to the dermatan sulfate, wherein the peptides comprise amino
acids 1-18 (RRANAALKAGELYKSILY) of SEQ ID NO: 1.
11. A composition comprising the peptidoglycan of claim 10 and a
pharmaceutically acceptable excipient or diluent, or a combination
thereof.
12. A synthetic peptidoglycan comprising heparin and from 1 to 10
collagen binding peptides, all of which are covalently bonded to
the heparin, wherein the peptides comprise amino acids 1-18
(RRANAALKAGELYKSILY) of SEQ ID NO: 1.
13. A composition comprising the peptidoglycan of claim 12 and a
pharmaceutically acceptable excipient or diluent, or a combination
thereof.
Description
SEQUENCE LISTING
The instant application contains a Sequence Listing which has been
submitted in ASCII format via EFS-Web and is hereby incorporated by
reference in its entirety. Said ASCII copy, created on Oct. 13,
2014, and is named 14466889_ST25.txt and is 4,613 bytes in
size.
TECHNICAL FIELD
This invention pertains to the field of collagen-binding synthetic
peptidoglycans and methods of forming and using the same.
BACKGROUND AND SUMMARY OF THE INVENTION
Collagen is the most abundant protein in the body, presenting many
biological signals and maintaining the mechanical integrity of many
different tissues. Its molecular organization determines its
function, which has made collagen fibrillogenesis a topic of
interest in many research fields. Collagen has the ability to
self-associate in vitro, forming gels that can act as a
3-dimensional substrate, and provide mechanical and biological
signals for cell growth. Research on collagen fibrillogenesis with
and without additional extracellular matrix components has raised
many questions about the interplay between collagen and other
extracellular matrix molecules. There are more than 20 types of
collagen currently identified, with type I being the most common.
Many tissues are composed primarily of type I collagen including
tendon, ligament, skin, and bone. While each of these structures
also contains other collagen types, proteoglycans and
glycosaminoglycans, and minerals in the case of bone, the principle
component is type I collagen. The dramatic difference in mechanical
integrity each of these structures exhibits is largely due to the
intricate organization of collagen and the interplay with other
non-collagen type I components.
Decorin is a proteoglycan that is known to influence collagen
fibrillogenesis, which consequently can modify the mechanical and
biological information in a collagen gel. The signals resulting
from structural changes in collagen organization, as well as the
unique signals contained in the glycosaminoglycan chains that are
part of proteoglycans, alter cellular behavior and offer a
mechanism to design collagen matrices to provide desired cellular
responses. Consequently, we have developed collagen-binding
synthetic peptidoglycans which influence collagen organization at
the molecular level. These collagen-binding synthetic
peptidoglycans are designed based on collagen binding peptides
attached to, for example, a glycan, such as a glycosaminoglycan or
a polysaccharide, and can be tailored with respect to these
components for specific applications. The collagen-binding
synthetic peptidoglycans described herein influence the
morphological, mechanical, and biological characteristics of
collagen matrices, and consequently alter cellular behavior, making
these molecules useful for tissue engineering applications.
In one embodiment, an engineered collagen matrix comprising a
collagen matrix and a collagen-binding synthetic peptidoglycan is
provided. In this embodiment, the 1) collagen can be crosslinked or
uncrosslinked, 2) the collagen-binding synthetic peptidoglycan can
have amino acid homology with a portion of the amino acid sequence
of a protein or a proteoglycan that regulates collagen
fibrillogenesis or the collagen-binding synthetic peptidoglycan can
be an aberrant collagen-binding synthetic peptidoglycan, 3) the
engineered collagen matrix can further comprise an exogenous
population of cells, 4) the exogenous population of cells can be
selected from non-keratinized or keratinized epithelial cells or a
population of cells selected from the group consisting of
endothelial cells, mesodermally derived cells, mesothelial cells,
synoviocytes, neural cells, glial cells, osteoblast cells,
fibroblasts, chondrocytes, tenocytes, smooth muscle cells, skeletal
muscle cells, cardiac muscle cells, multi-potential progenitor
cells (e.g., stem cells, including bone marrow progenitor cells),
and osteogenic cells, 5) the engineered collagen matrix can further
comprise at least one polysaccharide, 6) the collagen-binding
synthetic peptidoglycan can be a compound of formula P.sub.nG.sub.x
wherein n is 1 to 10, wherein x is 1 to 10, wherein P is a
synthetic peptide of about 5 to about 40 amino acids comprising a
sequence of a collagen-binding domain, and wherein G is a glycan
(e.g. a glycosaminoglycan or a polysaccharide), 7) the
collagen-binding synthetic peptidoglycan can be a compound of
formula (P.sub.nL).sub.xG wherein n is 1 to 5, wherein x is 1 to
10, wherein P is a synthetic peptide of about 5 to about 40 amino
acids comprising a sequence of a collagen-binding domain, wherein L
is a linker, and wherein G is a glycan, 8) the collagen-binding
synthetic peptidoglycan can be a compound of formula
P(LG.sub.n).sub.x wherein n is 1 to 5, wherein x is 1 to 10,
wherein P is a synthetic peptide of about 5 to about 40 amino acids
comprising a sequence of a collagen-binding domain, wherein L is a
linker, and wherein G is a glycan, 9) the synthetic peptide can
have amino acid homology with the amino acid sequence of a small
leucine-rich proteoglycan or a platelet receptor sequence, 10) the
synthetic peptide can have amino acid homology with the amino acid
sequence of a platelet collagen receptor sequence, 11) the peptide
can comprise an amino acid sequence selected from the group
consisting of RRANAALKAGELYKSILYGC [SEQ ID NO: 1], RLDGNEIKRGC [SEQ
ID NO: 2], AHEEISTTNEGVMGC [SEQ ID NO: 3],
NGVFKYRPRYFLYKHAYFYPPLKRFPVQGC [SEQ ID NO: 4], CQDSETRTFY [SEQ ID
NO: 5], TKKTLRTGC [SEQ ID NO: 6], GLRSKSKKFRRPDIQYPDATDEDITSHMGC
[SEQ ID NO: 7], SQNPVQPGC [SEQ ID NO: 8], SYIRIADTNITGC [SEQ ID NO:
9], SYIRIADTNIT [SEQ ID NO: 10], KELNLVYT [SEQ ID NO: 11],
KELNLVYTGC [SEQ ID NO: 12], GSITTIDVPWNV [SEQ ID NO: 13], and
GSITTIDVPWNVGC [SEQ ID NO: 14], 12) the glycan can be selected from
the group consisting of alginate, agarose, dextran, chondroitin,
dermatan, dermatan sulfate, heparan, heparin, keratin, and
hyaluronan, 13) the glycan can be selected from the group
consisting of dermatan sulfate, dextran, and heparin, 14) the
collagen can be selected from the group consisting of type I
collagen, type II collagen, type III collagen, type IV collagen,
and combinations thereof, 15) the glycan can be a glycosaminoglycan
or a polysaccharide, or 16) the invention can include any
combination of the features described in this paragraph.
In another illustrative embodiment, a method of preparing an
engineered collagen matrix is provided. The method comprises the
steps of providing a collagen solution, providing a
collagen-binding synthetic peptidoglycan, and polymerizing the
collagen in the presence of the collagen-binding synthetic
peptidoglycan to form the engineered collagen matrix. This
embodiment can include any of the features described in the
preceding paragraph. Also, in this embodiment, the amount of
collagen in the collagen solution can be from about 0.4 mg/mL to
about 6 mg/mL, and the molar ratio of the collagen to the
collagen-binding synthetic peptidoglycan can be from about 1:1 to
about 40:1.
In yet another embodiment a compound of formula P.sub.nG.sub.x is
provided wherein n is 1 to 10, wherein x is 1 to 10, wherein P is a
synthetic peptide of about 5 to about 40 amino acids comprising a
sequence of a collagen-binding domain, and wherein G is a
glycan.
In a further embodiment, a compound is provided of formula
(P.sub.nL).sub.xG wherein n is 1 to 5, wherein x is 1 to 10,
wherein P is a synthetic peptide of about 5 to about 40 amino acids
comprising a sequence of a collagen-binding domain, wherein L is a
linker, and G is a glycan.
In still another illustrative embodiment, a compound is provided of
formula P(LG.sub.n).sub.x wherein n is 1 to 5, wherein x is 1 to
10, wherein P is a synthetic peptide of about 5 to about 40 amino
acids comprising a sequence of a collagen-binding domain, wherein L
is a linker, and wherein G is a glycan. In any of these compound
embodiments the linker can comprise the formula
--SCH.sub.2CH.sub.2C(O)NHN.dbd., the glycan can be a
glycosaminoglycan or a polysaccharide, and any applicable features
described above can also be included.
In another aspect, a method of altering the structure or mechanical
characteristics of an engineered collagen matrix is provided. The
method comprises the steps of providing a collagen solution,
providing a collagen-binding synthetic peptidoglycan, and
polymerizing the collagen in the presence of the collagen-binding
synthetic peptidoglycan to form the altered, engineered collagen
matrix. Any applicable features described above can also be
included.
In another embodiment, a kit is provided. The kit can comprise any
of the engineered collagen matrices described above. In this
embodiment, the engineered collagen matrix can be sterilized, and
the kit can further comprise cells wherein the cells can be
selected from the group consisting of mesothelial cells,
synoviocytes, progenitor cells, fibroblasts, neural cells, glial
cells, osteoblast cells, chondrocytes, tenocytes, endothelial
cells, and smooth muscle cells. The engineered collagen matrix can
comprise any of the compounds described above.
In one embodiment, a method for inhibiting activation of platelets
is described, the method comprising the step of providing a
collagen-binding synthetic peptidoglycan for contacting collagen
wherein the collagen-binding synthetic peptidoglycan binds to the
collagen and wherein activation of the platelets is inhibited. In
another embodiment, a method for inhibiting adhesion of platelets
to collagen is described, the method comprising the step of
providing a collagen-binding synthetic peptidoglycan for contacting
collagen wherein the collagen-binding synthetic peptidoglycan binds
to the collagen, and wherein adhesion of the platelets to collagen
is inhibited. In another embodiment, either of the above methods
wherein the glycan is selected from the group consisting of
hyaluronan, heparin, and dextran is provided. In still another
embodiment, the collagen-binding synthetic peptidoglycan used in
any of the above methods comprises a peptide selected from the
group consisting of RRANAALKAGELYKSILYGC [SEQ ID NO: 1],
GSITTIDVPWNV [SEQ ID NO: 13], and GSITTIDVPWNVGC [SEQ ID NO:
14].
In yet another embodiment, a graft construct is provided. The graft
construct comprises any of the engineered collagen matrices
described above.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows a schematic representation of the interaction between
neighboring proteoglycans (referring to the gray bar having
triangles and labelled "GAG") on adjacent tropocollagen strands
which is important in determining the mechanical and alignment
properties of collagen matrices.
FIG. 2. AFM images made in contact mode, with a scan rate of 2 Hz
with Silicon Nitride contact mode tip k=0.05N/m tips and deflection
setpoint: 0-1 Volts, of gel samples prepared as in EXAMPLE 16 (10:1
collagen:treatment) after dehydration with ethanol. Samples are for
collagen alone (Collagen), and for collagen with dermatan sulfate
(DS), with decorin (Decorin), dermatan sulfate-RRANAALKAGELYKSILYGC
[SEQ ID NO: 1] conjugate (DS-SILY) and dermatan sulfate-SYIRIADTNIT
[SEQ ID NO: 10] conjugate (DS-SYIR).
FIG. 3. Surface Plasmon Resonance scan in association mode and
dissociation mode of peptide RRANAALKAGELYKSILYGC [SEQ ID NO: 1]
(SILY) binding to collagen bound to CM-3 plates. SILY was dissolved
in 1.times.HBS-EP buffer at varying concentrations from 100 .mu.M
to 1.5 .mu.m in 2-fold dilutions.
FIG. 4. Binding of dansyl-modified peptide SILY to collagen
measured in 96-well high-binding plate (black with a clear bottom
(Costar)). PBS, buffer only; BSA, BSA-treated well; Collagen,
collagen-treated well. Fluorescence readings were taken on an M5
Spectramax Spectrophotometer (Molecular Devices) at
excitation/emission wavelengths of 335 nm/490 nm, respectively.
FIG. 5. Collagen-dansyl-modified peptide SILY binding curve derived
from fluorescence data described in FIG. 4.
FIG. 6. A schematic description of the reagent,
3-(2-pyridyldithio)propionyl hydrazide (PDPH), and the chemistry of
the two-step conjugation of a cysteine-containing peptide with an
oxidized glycosylaminoglycoside showing the release of
2-pyridylthiol in the final step.
FIG. 7. Measurement of absorbance at 343 nm before DTT treatment of
oxidized dermatan sulfate conjugated to PDPH, and after treatment
with DTT, which releases 2-pyridylthiol from the conjugate. The
measurements allow determination of the ratio of PDPH to oxidized
dermatan sulfate. The measured .DELTA.A=0.35, corresponds to 1.1
PDPH molecules/DS.
FIG. 8. Binding of dansyl-modified peptide SILY conjugated to
dermatan sulfate as described herein to collagen measured in
96-well high-binding plate (black with a clear bottom (Costar)).
PBS, buffer only; BSA, BSA-treated well; Collagen, collagen-treated
well. Fluorescence readings were taken on an M5 Spectramax
Spectrophotometer (Molecular Devices) at excitation/emission
wavelengths of 335 nm/490 nm respectively.
FIG. 9. Measurement of Shear modulus of gel samples (4 mg/mL
collagen, 10:1 collagen:treatment) on a AR-G2 rheometer with 20 mm
stainless steel parallel plate geometry (TA Instruments, New
Castle, Del.), and the 20 mm stainless steel parallel plate
geometry was lowered to a gap distance of 600 .mu.m using a normal
force control of 0.25N. Col, no treatment, i.e. collagen alone;
Col+DS, collagen+dermatan sulfate; Col+decorin, collagen+decorin;
Col+DS-SYIR, collagen+dermatan sulfate-SYIR; Col+DS-SILY,
collagen+dermatan sulfate-SILY conjugate; Col+SILY, collagen+SILY
peptide; Col+SYIR, collagen+SYIRIADTNIT [SEQ ID NO: 10] (SYIR)
peptide.
FIG. 10. Measurement of Shear modulus of gel samples (4 mg/mL
collagen, 5:1 collagen:treatment) on a AR-G2 rheometer with 20 mm
stainless steel parallel plate geometry (TA Instruments, New
Castle, Del.), and the 20 mm stainless steel parallel plate
geometry was lowered to a gap distance of 600 .mu.m using a normal
force control of 0.25N. Col, no treatment, i.e. collagen alone;
Col+DS, collagen+dermatan sulfate; Col+decorin, collagen+decorin;
Col+DS-SYIR, collagen+dermatan sulfate-SYIR; Col+DS-SILY,
collagen+dermatan sulfate-SILY conjugate; Col+SILY, SILY peptide;
Col+SYIR, collagen+SYIR peptide.
FIG. 11. Measurement of Shear modulus of gel samples (4 mg/mL
collagen, 30:1 collagen:treatment) on a AR-G2 rheometer with 20 mm
stainless steel parallel plate geometry (TA Instruments, New
Castle, Del.), and the 20 mm stainless steel parallel plate
geometry was lowered to a gap distance of 600 .mu.m using a normal
force control of 0.25N. Col, no treatment, i.e. collagen alone;
Col+DS, collagen+dermatan sulfate; Col+decorin, collagen+decorin;
Col+DS-SYIR, collagen+dermatan sulfate-SYIR conjugate; Col+DS-SILY,
collagen+dermatan sulfate-SILY conjugate; Col+SILY, collagen+SILY
peptide; Col+SYIR, collagen+SYIR peptide.
FIG. 12. Measurement of Shear modulus of gel samples (1.5 mg/mL
collagen III, 5:1 collagen:treatment) on a AR-G2 rheometer with 20
mm stainless steel parallel plate geometry (TA Instruments, New
Castle, Del.), and the 20 mm stainless steel parallel plate
geometry was lowered to a gap distance of 500 .mu.m using a normal
force control of 0.25N. .diamond-solid.--no treatment, i.e.
collagen III alone; .box-solid.--collagen+dermatan sulfate
(1:1);+--collagen+dermatan sulfate (5:1); x--collagen+dermatan
sulfate-KELNLVYTGC [SEQ ID NO: 12] (DS-KELN) conjugate (1:1);
.tangle-solidup.--collagen+dermatan sulfate-KELN conjugate (5:1);
.circle-solid.--collagen+KELNLVYTGC [SEQ ID NO: 12] (KELN)
peptide.
FIG. 13. Measurement of Shear modulus of gel samples (1.5 mg/mL
collagen III, 5:1 collagen treatment) on a AR-G2 rheometer with 20
mm stainless steel parallel plate geometry (TA Instruments, New
Castle, Del.), and the 20 mm stainless steel parallel plate
geometry was lowered to a gap distance of 500 .mu.m using a normal
force control of 0.25N. .diamond-solid.--no treatment, i.e.
collagen III alone; .box-solid.--collagen+dermatan sulfate
(1:1);+--collagen+dermatan sulfate (5:1);
.times.--collagen+dermatan sulfate-GSIT conjugate (DS-GSIT) (1:1);
.tangle-solidup.--collagen+dermatan sulfate-GSIT conjugate (5:1);
.circle-solid.--collagen+GSITTIDVPWNVGC [SEQ ID NO: 14] (GSIT)
peptide.
FIG. 14. Turbidity measurement. Gel solutions were prepared as
described in EXAMPLE 16 (collagen 4 mg/mL and 10:1 collagen to
treatment, unless otherwise indicated) and 50 .mu.L/well were added
at 4.degree. C. to a 384-well plate. The plate was kept at
4.degree. C. for 4 hours before initiating fibril formation. A
SpectraMax M5 at 37.degree. C. was used to measure absorbance at
313 nm at 30 s intervals for 6 hours. Col, no treatment, i.e.,
collagen alone; DS, collagen+dermatan sulfate; decorin,
collagen+decorin; DS-SILY, collagen+dermatan sulfate-SILY
conjugate; DS-SYIR, collagen+dermatan sulfate-SYIR conjugate.
FIG. 15. Turbidity measurement. Gel solutions were prepared as
described in EXAMPLE 16 (collagen 4 mg/mL and 10:1 collagen to
treatment, unless otherwise indicated) and 50 .mu.L/well were added
at 4.degree. C. to a 384-well plate. The plate was kept at
4.degree. C. for 4 hours before initiating fibril formation. A
SpectraMax M5 at 37.degree. C. was used to measure absorbance at
313 nm at 30 s intervals for 6 hours. Col, no treatment, i.e.,
collagen alone; DS, collagen+dermatan sulfate; decorin,
collagen+decorin; DS-SILY, collagen+dermatan sulfate-SILY
conjugate.
FIG. 16. Turbidity measurement. Gel solutions were prepared as
described in EXAMPLE 16 (collagen 4 mg/mL and 1:1 collagen to
treatment, unless otherwise indicated) and 50 .mu.L/well were added
at 4.degree. C. to a 384-well plate. The plate was kept at
4.degree. C. for 4 hours before initiating fibril formation. A
SpectraMax M5 at 37.degree. C. was used to measure absorbance at
313 nm at 30 s intervals for 6 hours. Col, no treatment, i.e.,
collagen alone; DS, collagen+dermatan sulfate 10:1; SILY,
collagen+SILY peptide; SYIR, collagen+SYIR peptide.
FIG. 17. Half-life of fibrillogenesis measured from the data
presented in FIG. 14. Col, no treatment, i.e., collagen alone; DS,
collagen+dermatan sulfate; decorin, collagen+decorin; DS-SILY,
collagen+dermatan sulfate-SILY conjugate; DS-SYIR,
collagen+dermatan sulfate-SYIR conjugate.
FIG. 18. Confocal Reflection Microscopy images of gels prepared
according to EXAMPLE 16 (4 mg/mL collagen, 10:1 collagen:treatment)
recorded with an Olympus FV1000 confocal microscope using a
60.times., 1.4 NA water immersion lens. Samples were illuminated
with 488 nm laser light and the reflected light was detected with a
photomultiplier tube using a blue reflection filter. Each gel was
imaged 100 .mu.M from the bottom of the gel, and three separate
locations were imaged to ensure representative sampling. Collagen,
no treatment, i.e., collagen alone; Col+DS, collagen+dermatan
sulfate; Col+Decorin, collagen+decorin; Col+DS-SILY,
collagen+dermatan sulfate-SILY conjugate; Col+DS-SYIR,
collagen+dermatan sulfate-SYIR conjugate.
FIG. 19. Cryo-Scanning Electron Microscopy images of gel structure
at a magnification of 5000. Gels for cryo-SEM were formed, as in
EXAMPLE 16 (4 mg/mL collagen, 10:1 collagen:treatment), directly on
the SEM stage and incubated at 37.degree. C. overnight. Each sample
evaporated under sublimation conditions for 20 min. The sample was
coated by platinum sputter coating for 120 s. Samples were
transferred to the cryo-stage at -130.degree. C. and regions with
similar orientation were imaged for comparison across treatments.
Collagen, no treatment, i.e., collagen alone; Col+DS,
collagen+dermatan sulfate; Col+Decorin, collagen+decorin;
Col+DS-SILY, collagen+dermatan sulfate-SILY conjugate; Col+DS-SYIR,
collagen+dermatan sulfate-SYIR conjugate.
FIG. 20. Cryo-Scanning Electron Microscopy images of gel structure
at a magnification of 5000. Gels for cryo-SEM were formed, as
described in EXAMPLE 22 (1 mg/mL collagen (Type III), 1:1
collagen:treatment), directly on the SEM stage. Regions with
similar orientation were imaged for comparison across treatments.
Panel a, Collagen, no treatment, i.e., collagen alone; Panel b,
collagen+dermatan sulfate; Panel c, collagen+dermatan sulfate-KELN
conjugate; Panel d, collagen+dermatan sulfate-GSIT conjugate.
FIG. 21. The average void space fraction measured from the Cryo-SEM
images shown in FIG. 20. a) Collagen, no treatment, i.e., collagen
alone; b) collagen+dermatan sulfate; c) collagen+dermatan
sulfate-KELN conjugate; d) collagen+dermatan sulfate-GSIT
conjugate. All differences are significant with p=0.05.
FIG. 22. The average fibril diameter measured from the Cryo-SEM
images shown in FIG. 19. Collagen, no treatment, i.e., collagen
alone; Col+DS, collagen+dermatan sulfate; Col+Decorin,
collagen+decorin; Col+DS-SILY, collagen+dermatan sulfate-SILY
conjugate; Col+DS-SYIR, collagen+dermatan sulfate-SYIR
conjugate.
FIG. 23. The average distance between collagen sheets measured from
the Cryo-SEM images shown in FIG. 19. Collagen, no treatment, i.e.,
collagen alone; Col+DS, collagen+dermatan sulfate; Col+Decorin,
collagen+decorin; Col+DS-SILY, collagen+dermatan sulfate-SILY
conjugate; Col+DS-SYIR, collagen+dermatan sulfate-SYIR
conjugate.
FIG. 24. Measurement of absorbance at 343 nm before treatment of
oxidized heparin conjugated to PDPH, and after treatment with SILY,
which releases 2-pyridylthiol from the conjugate and allows
determination of the ratio of SILY peptide conjugated to oxidized
heparin. The measured .DELTA.A, corresponds to 5.44 SILY
molecules/oxidized heparin.
FIG. 25. Measuring Human Coronary Artery Smooth Muscle Cell
Proliferation in Collagen Gels Prepared with Collagen-binding
synthetic peptidoglycans. Collagen, no treatment, i.e., collagen
alone; DS, collagen+dermatan sulfate; DS-SILY, collagen+dermatan
sulfate-SILY conjugate; DS-SYIR, collagen+dermatan sulfate-SYIR
conjugate; SILY, collagen+SILY peptide; and SYIR, collagen+SYIR
peptide.
FIG. 26. DS-SILY Conjugation Characterization. After 2 hours, a
final .DELTA.A.sub.343nm corresponded to 1.06 SILY molecules added
to each DS molecule. Note, t=0 is an approximate zero time point
due to the slight delay between addition of SILY to the DS-PDPH and
measurement of the solution at 343 nm.
FIG. 27. Conjugation of Dc13 to DS. Production of pyridine-2-thione
measured by an increase in absorbance at 343 nm indicates 0.99 Dc13
peptides per DS polymer chain.
FIG. 28. Microplate Fluorescence Binding of DS-ZDc13 to Collagen.
DS-ZDc13 bound specifically to the collagen surface in a
dose-dependent manner.
FIG. 29. Collagen Fibrillogenesis by Turbidity Measurements.
DS-Dc13 delays fibrillogenesis and decreases overall absorbance in
a dose-dependent manner. Free Dc13 peptide, in contrast, appears to
have little effect on fibrillogenesis compared to collagen alone at
the high 1:1 collagen:additive molar ratio.
FIG. 30. Average Fibril Diameter from Cryo-SEM. A. Decorin and
synthetic peptidoglycans significantly decrease fibril diameter
over collagen or collagen+DS. B. Compared to collagen alone, free
peptide Dc13 does not affect fibril diameter while SILY results in
a decrease in fibril diameter.
FIG. 31. Gel Compaction. A. and B. Days 3 and 5 respectively:
Decorin and peptidoglycans are significant relative to collagen and
DS, * indicates DS-Dc13 and DS are not significant at day 3. Bars
indicate no significance. C. Day 7: +Decorin is significant against
all samples, # DS is significant compared to collagen. D. Day 10:
++ collagen and DS are significant, DS-Dc13 is significant compared
to decorin and collagen.
FIG. 32. Elastin Estimate by Fastin Assay. A. DS-SILY significantly
increased elastin production over all samples. DS and DS-Dc13
significantly decreased elastin production over collagen. Control
samples of collagen gels with no cells showed no elastin
production. B. Free peptides resulted in a slight decrease in
elastin production compared to collagen, but no points were
significant.
FIG. 33. SEM Images of Platelet-Rich Plasma Incubated Slides.
Arrows in Heparin-SILY treatment indicate fibril-like structures
unique to this treatment. Scale bar=100 .mu.m.
FIG. 34. Fibril Density from Cryo-SEM. Fibril density, defined as
the ratio of fibril containing area to void space. DS-SILY and free
SILY peptide had significantly greater fibril density, while
collagen had significantly lower fibril density. DS-Dc13 was not
significant compared to collagen.
FIG. 35. Storage Modulus (G') of Collagen Gels. Rheological
mechanical testing of collagen gels formed with each additive at A.
5:1 B. 10:1 and C. 30:1 molar ratio of collagen:additive. Frequency
sweeps from 0.1 Hz to 1.0 Hz with a controlled stress of 1.0 Pa
were performed. G'avg.+-.S.E. are presented.
FIG. 36. Cell Proliferation and Cytotoxicity Assays. No significant
differences were found between all additives in A. CyQuant B. Live
and C. Dead assays.
FIG. 37. Cryo-SEM Images for Fibril Density. Collagen gels formed
in the presence of each additive at a 10:1 molar ratio of
collagen:additive. A. DS, Decorin, or peptidoglycans. B. Free
Peptides. Images are taken at 10,000.times., Scale bar=5 .mu.m.
FIG. 38. AFM Images of Collagen Gels. Collagen gels were formed in
the presence of each additive at a 10:1 molar ratio of
collagen:additive. D-banding is observed for all additives. Images
are 1 .mu.m.sup.2.
FIG. 39. Inhibition of Platelet Activation. Measured by determining
the release of activation factors Platelet Factor 4 (PF-4) and
.beta.-thromboglobulin (Nap-2). Collagen immobilized on the surface
of a 96-well plate was pre-incubated with each treatment and
subsequently incubated with platelet rich plasma (PRP). Values are
reported as a percentage of activation factor released by the
treatment compared to the amount of activation factor released by
the control treatment (phosphate buffered saline, PBS). The *
indicates that the difference is significant vs. collagen surface
with no treatment (phosphate buffered saline, PBS). Dex, dextran;
Dex-SILY9, dextran-(SILY).sub.9 conjugate; Hep, heparin; Hep-SILY,
heparin-SILY conjugate; HA, hyaluronan; HA-SILY, hyaluronan-SILY
conjugate; SILY, SILY peptide. Due to solubility limits, Hep,
Hep-SILY, HA, and HA-SILY were incubated at 25 .mu.M. All other
treatments were at 50 .mu.M (after the treatment was removed, the
plates were washed with PBS<1 min, before addition of PRP). Hep
and HA (hyaluronic acid) conjugates contained approximately 4
peptides per polysaccharide.
FIG. 40. Inhibition of Platelet Activation. Measured by determining
the release of activation factors Platelet Factor 4 (PF-4) and
.beta.-thromboglobulin (Nap-2). Collagen immobilized on the surface
of a 96-well plate was pre-incubated with each treatment and
subsequently incubated with platelet rich plasma (PRP). Values are
reported as a percentage of activation factor released by the
treatment compared to the amount of activation factor released by
the control treatment (phosphate buffered saline, PBS). Dex,
dextran; Dex-SILY6, dextran-(SILY).sub.6 conjugate; Hep, heparin;
Hep-GSIT, heparin-GSIT conjugate; GSIT, GSIT peptide; SILY, SILY
peptide. The values measured for all treatments are significant vs.
PBS. Dex, SILY, and Dex-SILY6 are at 25 .mu.M, all other treatments
are at 50 .mu.M. The ** indicates that the value for the Hep-GSIT
treatment was significant vs. the values for the Hep treatment,
similarly the value for the Dex-SILY6 treatment was significant vs.
the value for the Dex treatment for PF4. (After the treatment was
removed the plates were rinsed for 20 min). Hep conjugates
contained approximately 4 peptides per polysaccharide.
FIG. 41. Inhibition of Platelet Binding to Collagen by Colorimetric
Assay. Collagen immobilized on the surface of a 96-well plate was
pre-incubated with each treatment and subsequently incubated with
platelet rich plasma (PRP). Microplate assay prepared as described
was pre-incubated with treatments Collagen, PBS only; Dextran;
Dex-SILY6, dextran-(SILY).sub.6; SILY, SILY peptide. * Significant
vs. collagen (no treatment).
FIG. 42. Fluorescence image of adhered platelets. Adhered platelets
were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton
X-100, and platelet actin was labeled with phalloidin-AlexaFluor
488. The adhered platelets were imaged using an upright fluorescent
microscope using a DAPI filter. No treatment, i.e. collagen treated
with PBS.
FIG. 43. Fluorescence image of adhered platelets. Adhered platelets
were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton
X-100, and platelet actin was labeled with phalloidin-AlexaFluor
488. The adhered platelets were imaged using an upright
fluorescence microscope using a DAPI filter. Treatment:
dextran.
FIG. 44. Fluorescence image of adhered platelets. Adhered platelets
were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton
X-100, and platelet actin was labeled with phalloidin-AlexaFluor
488. The adhered platelets were imaged using an upright
fluorescence microscope using a DAPI filter. Treatment:
dextran-SILY9 conjugate.
FIG. 45. Fluorescence image of adhered platelets. Adhered platelets
were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton
X-100, and platelet actin was labeled with phalloidin-AlexaFluor
488. The adhered platelets were imaged using an upright
fluorescence microscope using a DAPI filter. No treatment, i.e.
collagen treated with PBS.
FIG. 46. Fluorescence image of adhered platelets. Adhered platelets
were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton
X-100, and platelet actin was labeled with phalloidin-AlexaFluor
488. The adhered platelets were imaged using an upright
fluorescence microscope using a DAPI filter. Treatment:
hyaluronan.
FIG. 47. Fluorescence image of adhered platelets. Adhered platelets
were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton
X-100, and platelet actin was labeled with phalloidin-AlexaFluor
488. The adhered platelets were imaged using an upright
fluorescence microscope using a DAPI filter. Treatment:
hyaluronan-SILY conjugate.
FIG. 48. Fluorescence image of adhered platelets. Adhered platelets
were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton
X-100, and platelet actin was labeled with phalloidin-AlexaFluor
488. The adhered platelets were imaged using an upright
fluorescence microscope using a DAPI filter. No treatment, i.e.
collagen treated with PBS.
FIG. 49. Fluorescence image of adhered platelets. Adhered platelets
were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton
X-100, and platelet actin was labeled with phalloidin-AlexaFluor
488. The adhered platelets were imaged using an upright
fluorescence microscope using a DAPI filter. Treatment:
heparin.
FIG. 50. Fluorescence image of adhered platelets. Adhered platelets
were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton
X-100, and platelet actin was labeled with phalloidin-AlexaFluor
488. The adhered platelets were imaged using an upright
fluorescence microscope using a DAPI filter. Treatment:
heparin-SILY conjugate.
FIG. 51. Fluorescence image of adhered platelets. Adhered platelets
were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton
X-100, and platelet actin was labeled with phalloidin-AlexaFluor
488. The adhered platelets were imaged using an upright
fluorescence microscope using a DAPI filter. No treatment, i.e.
collagen treated with PBS.
FIG. 52. Fluorescence image of adhered platelets. Adhered platelets
were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton
X-100, and platelet actin was labeled with phalloidin-AlexaFluor
488. The adhered platelets were imaged using an upright
fluorescence microscope using a DAPI filter. Treatment: SILY
peptide.
FIG. 53. Collagen Degradation Determined by Hydroxyproline.
Treatments: Ctrl, no cells added; Col, collagen without added
treatment; DS, dermatan sulfate; Decorin; DS-SILY, dermatan
sulfate-SILY conjugate; DS-Dc13, dermatan sulfate-Dc13 conjugate;
SILY, SILY peptide; Dc13, Dc13 peptide.
FIG. 54. Inhibition of Platelet Activation. Measured by determining
the release of activation factors Platelet Factor 4 (PF-4) and
.beta.-thromboglobulin (Nap-2). Type I and III collagen gels on the
surface of a 96-well plate were pre-incubated with each treatment
and subsequently incubated with PRP. Platelet activation was
measured by the release of activation factors PF-4 and Nap-2.
Treatments: PBS, buffer alone; Dex, dextran; Dex-SILY, dextran-SILY
conjugate; Dex-GSIT, dextran-GSIT conjugate; Dex-KELN, dextran-KELN
conjugate; Dex-Dc13, dextran-Dc13 conjugate; SILY, SILY peptide;
GSIT, GSIT peptide; KELN, KELN peptide; Dc13, Dc13 peptide;
Dex-SILY+Dex-GSIT; combination of dextran-SILY conjugate and
dextran-GSIT conjugate; SILY+GSIT; combination of SILY peptide and
GSIT peptide. * Indicates the results are significant vs. collagen
surface with no treatment (PBS). ** Indicates the results are also
significant vs. collagen surface with Dex. *** Indicates the
results are also significant vs. collagen surface with
corresponding peptide control. All peptidoglycans caused
significant decrease in NAP-2 release compared to no treatment
(PBS) or dextran treatment, while Dex-GSIT additionally decreased
release over its peptide control (GSIT). Dex-GSIT and Dex-KELN
significantly decreased PF-4 release relative to no treatment (PBS)
and dextran treatment, while Dex-Dc13 significantly decreased PF-4
release over no treatment (PBS).
FIG. 55. Inhibition of Platelet Binding to Collagen (Adhesion) by
Colorimetric Assay. Treatments: PBS, buffer alone; Dex, dextran;
Dex-SILY, dextran-SILY conjugate; Dex-GSIT, dextran-GSIT conjugate;
Dex-KELN, dextran-KELN conjugate; Dex-Dc13, dextran-Dc13 conjugate;
SILY, SILY peptide; GSIT, GSIT peptide; KELN, KELN peptide; Dc13,
Dc13 peptide; Dex-SILY+Dex-GSIT; combination of dextran-SILY
conjugate and dextran-GSIT conjugate; SILY+GSIT; combination of
SILY peptide and GSIT peptide. * Significant vs. Collagen surface
with no treatment (PBS). ** Also significant vs. collagen surface
with Dex. *** Also significant vs. collagen surface with
corresponding peptide control. Dex-SILY and Dex-KELN had
significantly decreased platelet adherence as compared to no
treatment (PBS) or Dextran treatment, while Dex-GSIT additionally
decreased platelet adherence over its peptide control treatment
(GSIT).
DETAILED DESCRIPTION OF THE ILLUSTRATIVE EMBODIMENTS
As used in accordance with this invention, a "collagen-binding
synthetic peptidoglycan" means a collagen-binding conjugate of a
glycan with a synthetic peptide. The "collagen-binding synthetic
peptidoglycans" can have amino acid homology with a portion of a
protein or a proteoglycan not normally involved in collagen
fibrillogenesis. These collagen-binding synthetic peptidoglycans
are referred to herein as "aberrant collagen-binding synthetic
peptidoglycans". The aberrant collagen-binding synthetic
peptidoglycans may or may not affect collagen fibrillogenesis.
Other collagen-binding synthetic peptidoglycans can have amino acid
homology to a portion of a protein or to a proteoglycan normally
involved in collagen fibrillogenesis. These collagen-binding
synthetic peptidoglycans are referred to herein as "fibrillogenic
collagen-binding synthetic peptidoglycans".
As used herein an "engineered collagen matrix" means a collagen
matrix where the collagen is polymerized in vitro in combination
with a collagen-binding synthetic peptidoglycan under predetermined
conditions that can be varied and are selected from the group
consisting of, but not limited to, pH, phosphate concentration,
temperature, buffer composition, ionic strength, and composition
and concentration of the collagen.
As used herein an "engineered graft construct" means a graft
construct comprising an "engineered collagen matrix."
In one aspect of the invention, an engineered collagen matrix is
provided. The engineered collagen matrix comprises collagen and a
collagen-binding synthetic peptidoglycan. In one embodiment, the
engineered collagen matrix may be uncrosslinked. In another
embodiment, the matrix may be crosslinked. In various illustrative
embodiments, crosslinking agents, such as carbodiimides, aldehydes,
lysl-oxidase, N-hydroxysuccinimide esters, imidoesters, hydrazides,
and maleimides, as well as various natural crosslinking agents,
including genipin, and the like, can be added before, during, or
after polymerization of the collagen in solution.
In various illustrative embodiments, the collagen used herein to
prepare an engineered collagen matrix may be any type of collagen,
including collagen types I to XXVIII, alone or in any combination,
for example, collagen types I, II, III, and/or IV may be used. In
one embodiment, the engineered collagen matrix is formed using
commercially available collagen (e.g., Sigma, St. Louis, Mo.). In
an alternative embodiment, the collagen can be purified from
submucosa-containing tissue material such as intestinal, urinary
bladder, or stomach tissue. In a further embodiment, the collagen
can be purified from tail tendon. In an additional embodiment, the
collagen can be purified from skin. In various aspects, the
collagen can also contain endogenous or exogenously added
non-collagenous proteins in addition to the collagen-binding
synthetic peptidoglycans, such as fibronectin or silk proteins,
glycoproteins, and polysaccharides, or the like. The engineered
graft constructs or engineered collagen matrices prepared by the
methods described herein can serve as constructs for the regrowth
of endogenous tissues at the implantation site (e.g., biological
remodeling) which can assume the characterizing features of the
tissue(s) with which they are associated at the site of
implantation or injection.
In various illustrative aspects, the collagen-binding synthetic
peptidoglycans used to form the engineered graft constructs or
engineered collagen matrices in accordance with the invention
comprise synthetic peptides of about 5 to about 40 amino acids. In
some embodiments, these peptides have homology to the amino acid
sequence of a small leucine-rich proteoglycan or a platelet
receptor sequence. In various embodiments the synthetic peptide
comprises an amino acid sequence selected from the group consisting
of RRANAALKAGELYKSILYGC [SEQ ID NO: 1], RLDGNEIKRGC [SEQ ID NO: 2],
AHEEISTTNEGVMGC [SEQ ID NO: 3], NGVFKYRPRYFLYKHAYFYPPLKRFPVQGC [SEQ
ID NO: 4], CQDSETRTFY [SEQ ID NO: 5], TKKTLRTGC [SEQ ID NO: 6],
GLRSKSKKFRRPDIQYPDATDEDITSHMGC [SEQ ID NO: 7], SQNPVQPGC [SEQ ID
NO: 8], SYIRIADTNITGC [SEQ ID NO: 9], SYIRIADTNIT [SEQ ID NO: 10],
KELNLVYT [SEQ ID NO: 11], KELNLVYTGC [SEQ ID NO: 12], GSITTIDVPWNV
[SEQ ID NO: 13], and GSITTIDVPWNVGC [SEQ ID NO: 14]. In another
embodiment, the synthetic peptide can comprise or can be an amino
acid sequence selected from the group consisting of
RRANAALKAGELYKSILYGC [SEQ ID NO: 1], RLDGNEIKRGC [SEQ ID NO: 2],
AHEEISTTNEGVMGC [SEQ ID NO: 3], NGVFKYRPRYFLYKHAYFYPPLKRFPVQGC [SEQ
ID NO: 4], CQDSETRTFY [SEQ ID NO: 5], TKKTLRTGC [SEQ ID NO: 6],
GLRSKSKKFRRPDIQYPDATDEDITSHMGC [SEQ ID NO: 7], SQNPVQPGC [SEQ ID
NO: 8], SYIRIADTNITGC [SEQ ID NO: 9], SYIRIADTNIT [SEQ ID NO: 10],
KELNLVYT [SEQ ID NO: 11], KELNLVYTGC [SEQ ID NO: 12], GSITTIDVPWNV
[SEQ ID NO: 13], GSITTIDVPWNVGC [SEQ ID NO: 14], and an amino acid
sequence with 80%, 85%, 90%, 95%, or 98% homology with to any of
these fourteen amino acid sequences. The synthetic peptide can also
be any peptide of 5 to 40 amino acids selected from peptides that
have collagen-binding activity and that are 80%, 85%, 90%, 95%,
98%, or 100% homologous with the collagen-binding domain(s) of the
von Willebrand factor or a platelet collagen receptor as described
in Chiang, et al., J. Biol. Chem. 277: 34896-34901 (2002),
Huizinga, et al., Structure 5: 1147-1156 (1997), Romijn, et al., J.
Biol. Chem. 278: 15035-15039 (2003), and Chiang, et al., Cardio.
& Haemato. Disorders-Drug Targets 7: 71-75 (2007), each
incorporated herein by reference.
The glycan (e.g. glycosaminoglycan, abbreviated GAG, or
polysaccharide) attached to the synthetic peptide can be selected
from the group consisting alginate, agarose, dextran, chondroitin,
dermatan, dermatan sulfate, heparan, heparin, keratin, and
hyaluronan. In one embodiment, the glycan is selected from the
group consisting of dermatan sulfate, dextran, and heparin.
In one illustrative aspect, the engineered collagen matrix or the
engineered graft construct may be sterilized. As used herein
"sterilization" or "sterilize" or "sterilized" means disinfecting
the matrix or graft construct by removing unwanted contaminants
including, but not limited to, endotoxins, nucleic acid
contaminants, and infectious agents.
In various illustrative embodiments, the engineered collagen matrix
or engineered graft construct can be disinfected and/or sterilized
using conventional sterilization techniques including
glutaraldehyde tanning, formaldehyde tanning at acidic pH,
propylene oxide or ethylene oxide treatment, gas plasma
sterilization, gamma radiation, electron beam, and/or sterilization
with a peracid, such as peracetic acid. Sterilization techniques
which do not adversely affect the structure and biotropic
properties of the matrix or construct can be used. Illustrative
sterilization techniques are exposing the engineered graft
construct or engineered collagen matrix, to peracetic acid, 1-4
Mrads gamma irradiation (or 1-2.5 Mrads of gamma irradiation),
ethylene oxide treatment, or gas plasma sterilization. In one
embodiment, the engineered graft construct can be subjected to one
or more sterilization processes. In illustrative embodiments, the
collagen in solution can also be sterilized or disinfected. The
engineered collagen matrix or engineered graft construct may be
wrapped in any type of container including a plastic wrap or a foil
wrap, and may be further sterilized.
In any of these embodiments the engineered graft construct or
engineered collagen matrix may further comprise an added population
of cells. The added population of cells may comprise one or more
cell populations. In various embodiments, the cell populations
comprise a population of non-keratinized or keratinized epithelial
cells or a population of cells selected from the group consisting
of endothelial cells, mesodermally derived cells, mesothelial
cells, synoviocytes, neural cells, glial cells, osteoblasts,
fibroblasts, chondrocytes, tenocytes, smooth muscle cells, skeletal
muscle cells, cardiac muscle cells, multi-potential progenitor
cells (e.g., stem cells, including bone marrow progenitor cells),
and osteogenic cells. In various embodiments, the engineered graft
construct or engineered collagen matrix can be seeded with one or
more cell types in combination.
In various aspects, the engineered collagen matrices or engineered
graft constructs of the present invention can be combined with
nutrients, including minerals, amino acids, sugars, peptides,
proteins, vitamins (such as ascorbic acid), or laminin,
fibronectin, hyaluronic acid, fibrin, elastin, or aggrecan, or
growth factors such as epidermal growth factor, platelet-derived
growth factor, transforming growth factor beta, or fibroblast
growth factor, and glucocorticoids such as dexamethasone or
viscoelastic altering agents, such as ionic and non-ionic water
soluble polymers; acrylic acid polymers; hydrophilic polymers such
as polyethylene oxides, polyoxyethylene-polyoxypropylene
copolymers, and polyvinylalcohol; cellulosic polymers and
cellulosic polymer derivatives such as hydroxypropyl cellulose,
hydroxyethyl cellulose, hydroxypropyl methylcellulose,
hydroxypropyl methylcellulose phthalate, methyl cellulose,
carboxymethyl cellulose, and etherified cellulose; poly(lactic
acid), poly(glycolic acid), copolymers of lactic and glycolic
acids, or other polymeric agents both natural and synthetic. In
other illustrative embodiments, cross-linking agents, such as
carbodiimides, aldehydes, lysl-oxidase, N-hydroxysuccinimide
esters, imidoesters, hydrazides, and maleimides, as well as natural
crosslinking agents, including genipin, and the like can be added
before, concurrent with, or after the addition of cells.
As discussed above, in accordance with one embodiment, cells can be
added to the engineered collagen matrices or engineered graft
constructs after polymerization of the collagen or during collagen
polymerization. The engineered collagen matrices comprising the
cells can be subsequently injected or implanted in a host for use
as engineered graft constructs. In another embodiment, the cells on
or within the engineered collagen matrices can be cultured in
vitro, for a predetermined length of time, to increase the cell
number or to induce desired remodeling prior to implantation or
injection into a host.
In accordance with one embodiment, a kit is provided comprising the
engineered collagen matrix or engineered graft construct. The kit
itself can be within a container of any type, and the kit can
contain instructions for use of the components of the kit. In one
embodiment, cells may constitute a component of the kit. In various
embodiments, the characteristics of the engineered collagen
matrices may vary. In various illustrative embodiments, the
engineered collagen matrix or engineered graft construct in the kit
may comprise various other components, including non-collagenous
proteins and polysaccharides, in addition to the collagen-binding
synthetic peptidoglycan(s). In one embodiment, the kit comprises a
vessel, vial, container, bag, or wrap, for example, containing an
engineered collagen matrix or an engineered graft construct. In
another embodiment, the kit comprises separate vessels (e.g., a
vial, container, bag, or wrap), each containing one of the
following components: a collagen solution or lyophilized collagen
and one or more types of collagen-binding synthetic peptidoglycans.
In another embodiment, the kit comprises separate vessels, each
containing one of the following components: a collagen solution or
lyophilized collagen, a buffer, and one or more types of
collagen-binding synthetic peptidoglycans. In any of these
embodiments, the kits can further comprise a buffer, a sterilizing
or disinfecting agent, non-collagenous proteins or polysaccharides,
and/or instructional materials describing methods for using the kit
reagents or describing methods for using the engineered collagen
matrices or the engineered graft construct. The kit can also
contain one or more types of collagen-binding synthetic
peptidoglycans for use as pharmacological agents in the absence of
an engineered collagen matrix or an engineered graft construct. In
this embodiment, the kit can be within a container of any type, and
the kit can contain instructions for use of the collagen-binding
synthetic peptidoglycans.
In yet another embodiment, the kit further comprises a container
(e.g. a flask, an ampule, a vial, a tube, or a bottle, for example)
of cells, including but not limited to, a population of
non-keratinized or keratinized epithelial cells or a population of
cells selected from the group consisting of endothelial cells,
mesodermally derived cells, mesothelial cells, synoviocytes, neural
cells, glial cells, osteoblasts, fibroblasts, chondrocytes,
tenocytes, smooth muscle cells, skeletal muscle cells, cardiac
muscle cells, multi-potential progenitor cells (e.g., stem cells,
including bone marrow progenitor cells), and osteogenic cells. In
another embodiment the cells may be present on a plate. In one
embodiment, one or more containers of cells may be included and the
kit may comprise one or more cell type and cell culture
reagents.
In one illustrative aspect, a method of preparing an engineered
collagen matrix is provided. The method comprises the steps of
providing a collagen solution, providing a collagen-binding
synthetic peptidoglycan, and polymerizing the collagen in the
presence of the collagen-binding synthetic peptidoglycan to form
the engineered collagen matrix. In various embodiments, the
collagen-binding synthetic peptidoglycan can be an aberrant
collagen-binding synthetic peptidoglycan or a fibrillogenic
collagen-binding synthetic peptidoglycan with amino acid homology
to a portion of the amino acid sequence of a proteoglycan that
normally regulates collagen fibrillogenesis.
In embodiments where the collagen-binding synthetic peptidoglycan
is an aberrant collagen-binding synthetic peptidoglycan or a
fibrillogenic collagen-binding synthetic peptidoglycan, a method of
altering the structure or mechanical characteristics of a collagen
matrix is provided. As used herein, "altering" means changing the
mechanical or structural characteristics of a collagen matrix
polymerized in vitro in the presence of the collagen-binding
synthetic peptidoglycan relative to that of a collagen matrix
polymerized in the absence of the collagen-binding synthetic
peptidoglycan. The method comprises the steps of providing a
collagen solution, providing a collagen-binding synthetic
peptidoglycan, and polymerizing the collagen in the presence of the
collagen-binding synthetic peptidoglycan (e.g., aberrant or
fibrillogenic collagen-binding synthetic peptidoglycan) to form the
altered collagen matrix.
In one illustrative embodiment, the collagen solution provided can
have a collagen concentration ranging from about 0.4 mg/ml to about
6 mg/ml. In various embodiments, the collagen concentration may
range from about 0.5 mg/ml to about 10 mg/ml, about 0.1 mg/ml to
about 6 mg/ml, about 0.5 mg/ml to about 3 mg/ml, about 1 mg/ml to
about 3 mg/ml, and about 2 mg/ml to about 4 mg/ml.
As discussed above, in various illustrative aspects, the
collagen-binding synthetic peptidoglycans used to form the
engineered graft constructs or engineered collagen matrices in
accordance with the invention comprise peptides of about 5 to about
40 amino acids with homology to the amino acid sequence of a small
leucine-rich proteoglycan or a platelet receptor sequence. In
various embodiments the synthetic peptide comprises an amino acid
sequence selected from the group consisting of RRANAALKAGELYKSILYGC
[SEQ ID NO: 1], RLDGNEIKRGC [SEQ ID NO: 2], AHEEISTTNEGVMGC [SEQ ID
NO: 3], CQDSETRTFY [SEQ ID NO: 5], TKKTLRTGC [SEQ ID NO: 6],
GLRSKSKKFRRPDIQYPDATDEDITSHMGC [SEQ ID NO: 7], SQNPVQPGC [SEQ ID
NO: 8], SYIRIADTNITGC [SEQ ID NO: 9], SYIRIADTNIT [SEQ ID NO: 10],
KELNLVYT [SEQ ID NO: 11], KELNLVYTGC [SEQ ID NO: 12], GSITTIDVPWNV
[SEQ ID NO: 13], NGVFKYRPRYFLYKHAYFYPPLKRFPVQGC [SEQ ID NO: 4], and
GSITTIDVPWNVGC [SEQ ID NO: 14]. In another embodiment, the
synthetic peptide can comprise or can be an amino acid sequence
selected from the group consisting of RRANAALKAGELYKSILYGC [SEQ ID
NO: 1], RLDGNEIKRGC [SEQ ID NO: 2], AHEEISTTNEGVMGC [SEQ ID NO: 3],
NGVFKYRPRYFLYKHAYFYPPLKRFPVQGC [SEQ ID NO: 4], CQDSETRTFY [SEQ ID
NO: 5], TKKTLRTGC [SEQ ID NO: 6], GLRSKSKKFRRPDIQYPDATDEDITSHMGC
[SEQ ID NO: 7], SQNPVQPGC [SEQ ID NO: 8], SYIRIADTNITGC [SEQ ID NO:
9], SYIRIADTNIT [SEQ ID NO: 10], KELNLVYT [SEQ ID NO: 11],
KELNLVYTGC [SEQ ID NO: 12], GSITTIDVPWNV [SEQ ID NO: 13],
GSITTIDVPWNVGC [SEQ ID NO: 14], and an amino acid sequence with
80%, 85%, 90%, 95%, or 98% homology to any of these fourteen amino
acid sequences. The synthetic peptide can also be any peptide of 5
to 40 amino acids selected from peptides that have collagen-binding
activity and that are 80%, 85%, 90%, 95%, 98%, or 100% homologous
to the collagen-binding domain(s) of the von Willebrand factor or a
platelet collagen receptor as described in Chiang, et al., J. Biol.
Chem. 277: 34896-34901 (2002), Huizinga, et al., Structure 5:
1147-1156 (1997), Romijn, et al., J. Biol. Chem. 278: 15035-15039
(2003), and Chiang, et al., Cardio. & Haemato. Disorders-Drug
Targets 7: 71-75 (2007), each incorporated herein by reference.
The glycan attached to the synthetic peptide can be selected from
the group consisting of alginate, agarose, dextran, chondroitin,
dermatan, dermatan sulfate, heparan, heparin, keratin, and
hyaluronan. In one embodiment, the glycan is selected from the
group consisting of dermatan sulfate, dextran, and heparin. The
collagen-binding synthetic peptidoglycan can be lyophilized prior
to polymerization, for example, in a buffer or in water or in an
acid, such as hydrochloric acid or acetic acid. In one illustrative
aspect, the molar ratio of the collagen to the collagen-binding
synthetic peptidoglycan can be from about 1:1 to about 40:1.
The polymerizing step can be performed under conditions that are
varied where the conditions are selected from the group consisting
of pH, phosphate concentration, temperature, buffer composition,
ionic strength, the specific components present, and the
concentration of the collagen or other components present. In one
illustrative aspect, the collagen or other components, including
the collagen-binding synthetic peptidoglycan, can be lyophilized
prior to polymerization. The collagen or other components can be
lyophilized in an acid, such as hydrochloric acid or acetic
acid.
In various illustrative embodiments, the polymerization reaction is
conducted in a buffered solution using any biologically compatible
buffer known to those skilled in the art. For example, the buffer
may be selected from the group consisting of phosphate buffer
saline (PBS), Tris (hydroxymethyl) aminomethane Hydrochloride
(Tris-HCl), 3-(N-Morpholino) Propanesulfonic Acid (MOPS),
piperazine-n,n'-bis (2-ethanesulfonic acid) (PIPES),
[n-(2-Acetamido)]-2-Aminoethanesulfonic Acid (ACES),
N-[2-hydroxyethyl] piperazine-N'-[2-ethanesulfonic acid] (HEPES),
and 1,3-bis[tris(Hydroxymethyl) methylamino]propane (Bis Tris
Propane). In one embodiment the buffer is PBS, Tris, or MOPS and in
one embodiment the buffer system is PBS.
In various illustrative embodiments, the polymerization step is
conducted at a pH selected from the range of about 5.0 to about 11,
and in one embodiment polymerization is conducted at a pH selected
from the range of about 6.0 to about 9.0, and in one embodiment
polymerization is conducted at a pH selected from the range of
about 6.5 to about 8.5, and in another embodiment the
polymerization step is conducted at a pH selected from the range of
about 7.0 to about 8.5, and in another embodiment the
polymerization step is conducted at a pH selected from the range of
about 7.3 to about 7.4.
In other illustrative aspects, the ionic strength of the buffered
solution is also regulated. In accordance with one embodiment, the
ionic strength of the buffer is selected from a range of about 0.05
to about 1.5 M, in another embodiment the ionic strength is
selected from a range of about 0.10 to about 0.90 M, in another
embodiment the ionic strength is selected from a range of about
0.14 to about 0.30 M and in another embodiment the ionic strength
is selected from a range of about 0.14 to about 0.17 M.
In still other illustrative embodiments, the polymerization step is
conducted at temperatures selected from the range of about
0.degree. C. to about 60.degree. C. In other embodiments, the
polymerization step is conducted at temperatures above 20.degree.
C., and typically the polymerization is conducted at a temperature
selected from the range of about 20.degree. C. to about 40.degree.
C., and more typically the temperature is selected from the range
of about 30.degree. C. to about 40.degree. C. In one illustrative
embodiment the polymerization is conducted at about 37.degree.
C.
In yet other embodiments, the phosphate concentration of the buffer
is varied. For example, in one embodiment, the phosphate
concentration is selected from a range of about 0.005 M to about
0.5 M. In another illustrative embodiment, the phosphate
concentration is selected from a range of about 0.01 M to about 0.2
M. In another embodiment, the phosphate concentration is selected
from a range of about 0.01 M to about 0.1 M. In another
illustrative embodiment, the phosphate concentration is selected
from a range of about 0.01 M to about 0.03 M.
The engineered collagen matrices, including collagen-binding
synthetic peptidoglycans, of the present invention can be combined,
prior to, during, or after polymerization, with nutrients,
including minerals, amino acids, sugars, peptides, proteins,
vitamins (such as ascorbic acid), or other compounds such as
laminin and fibronectin, hyaluronic acid, fibrin, elastin, and
aggrecan, or growth factors such as epidermal growth factor,
platelet-derived growth factor, transforming growth factor beta,
vascular endothelial growth factor, or fibroblast growth factor,
and glucocorticoids such as dexamethasone, or viscoelastic altering
agents, such as ionic and non-ionic water soluble polymers; acrylic
acid polymers; hydrophilic polymers such as polyethylene oxides,
polyoxyethylene-polyoxypropylene copolymers, and polyvinylalcohol;
cellulosic polymers and cellulosic polymer derivatives such as
hydroxypropyl cellulose, hydroxyethyl cellulose, hydroxypropyl
methylcellulose, hydroxypropyl methylcellulose phthalate, methyl
cellulose, carboxymethyl cellulose, and etherified cellulose;
poly(lactic acid), poly(glycolic acid), copolymers of lactic and
glycolic acids, or other polymeric agents both natural and
synthetic.
In accordance with one embodiment, cells can be added as the last
step prior to the polymerization or after polymerization of the
engineered collagen matrix. In other illustrative embodiments,
cross-linking agents, such as carbodiimides, aldehydes,
lysl-oxidase, N-hydroxysuccinimide esters, imidoesters, hydrazides,
and maleimides, and the like can be added before, during, or after
polymerization.
In one embodiment, the engineered collagen matrix is formed using
commercially available collagen (e.g., Sigma, St. Louis, Mo.). In
an alternative embodiment, the collagen can be purified from
submucosa-containing tissue material such as intestinal, urinary
bladder, or stomach tissue. In a further embodiment, the collagen
can be purified from tail tendon. In a further embodiment, the
collagen can be purified from skin.
In one embodiment, the collagen-binding synthetic peptidoglycans
with amino acid homology to a portion of the amino acid sequence of
a proteoglycan that normally regulates collagen fibrillogenesis or
with amino acid homology to a portion of a protein or a peptide
that does not normally regulate fibrillogenesis, can be used to
form an engineered collagen matrix with desired structural or
mechanical characteristics. In another embodiment, the aberrant
collagen-binding synthetic peptidoglycans or fibrillogenic
collagen-binding synthetic peptidoglycans can be used to form an
engineered collagen matrix with desired, but altered structure or
mechanical characteristics.
The desired structural, microstructural, nanostructural, or
mechanical characteristics can, illustratively, include fibril
length, fibril diameter, fibril density, fibril volume fraction,
fibril organization, 3-dimensional shape or form, and viscoelastic,
tensile, shear, or compressive behavior (e.g., failure stress,
failure strain, and modulus), permeability, degradation rate,
swelling, hydration properties (e.g., rate and swelling), and in
vivo tissue remodeling properties, and desired in vitro and in vivo
cell responses. The engineered graft constructs and engineered
collagen matrices described herein can have desirable
biocompatibility and in vitro and in vivo remodeling properties,
among other desirable properties.
As used herein, a "modulus" can be an elastic or linear modulus
(defined by the slope of the linear region of the stress-strain
curve obtained using conventional mechanical testing protocols;
i.e., stiffness), a compressive modulus, a complex modulus, or a
shear storage modulus.
As used herein, a "fibril volume fraction" is defined as the
percent area of the total area occupied by fibrils in a
cross-sectional surface of the matrix in 3 dimensions and "void
space fraction" is defined as the percent area of the total area
not occupied by fibrils in a cross-sectional surface of the matrix
in 3 dimensions.
The engineered collagen matrices described herein comprise collagen
fibrils which typically pack in a quarter-staggered pattern giving
the fibril a characteristic striated appearance or banding pattern
along its axis. In various illustrative embodiments, qualitative
and quantitative microstructural characteristics of the engineered
collagen matrices can be determined by scanning electron
microscopy, transmission electron microscopy, confocal microscopy,
second harmonic generation multi-photon microscopy. In another
embodiment, tensile, compressive and viscoelastic properties can be
determined by rheometry or tensile testing. All of these methods
are known in the art or are further described in the Examples
section of this application or in Roeder et al., J. Biomech. Eng.,
vol. 124, pp. 214-222 (2002), in Pizzo et al., J. Appl. Physiol.,
vol. 98, pp. 1-13 (2004), Fulzele et al., Eur. J. Pharm. Sci., vol.
20, pp. 53-61 (2003), Griffey et al., J. Biomed. Mater. Res., vol.
58, pp. 10-15 (2001), Hunt et al., Am. J. Surg., vol. 114, pp.
302-307 (1967), and Schilling et al., Surgery, vol. 46, pp. 702-710
(1959), incorporated herein by reference.
In any of the above-described engineered collagen matrix,
engineered graft construct, kit, or method embodiments, the
collagen-binding synthetic peptidoglycan can be a compound of any
of the following formulas P.sub.nG.sub.x wherein n is 1 to 10; A)
wherein x is 1 to 10; wherein P is a synthetic peptide of about 5
to about 40 amino acids comprising a sequence of a collagen-binding
domain; and wherein G is a glycan. OR (P.sub.nL).sub.xG wherein n
is 1 to 5; B) wherein x is 1 to 10; wherein P is a synthetic
peptide of about 5 to about 40 amino acids comprising a sequence of
a collagen-binding domain; wherein L is a linker; and wherein G is
a glycan. OR P(LG.sub.n).sub.x wherein n is 1 to 5; C) wherein x is
1 to 10; wherein P is a synthetic peptide of about 5 to about 40
amino acids comprising a sequence of a collagen-binding domain;
wherein L is a linker; and wherein G is a glycan.
In alternative embodiments, a compound of any of the following
formulas is provided P.sub.nG.sub.x wherein n is 1 to 10; A)
wherein x is 1 to 10; wherein P is a synthetic peptide of about 5
to about 40 amino acids comprising a sequence of a collagen-binding
domain; and wherein G is a glycan. OR (P.sub.nL).sub.xG wherein n
is 1 to 5; B) wherein x is 1 to 10; wherein P is a synthetic
peptide of about 5 to about 40 amino acids comprising a sequence of
a collagen-binding domain; wherein L is a linker; and wherein G is
a glycan. OR P(LG.sub.n).sub.x wherein n is 1 to 5; C) wherein x is
1 to 10; wherein P is a synthetic peptide of about 5 to about 40
amino acids comprising a sequence of a collagen-binding domain;
wherein L is a linker; and wherein G is a glycan.
In another embodiment, a collagen-binding synthetic peptidoglycan
comprising a synthetic peptide of about 5 to about 40 amino acids
with amino acid sequence homology to a collagen binding peptide
(e.g. a portion of an amino acid sequence of a collagen binding
protein or proteoglycan) conjugated to heparin, dextran, or
hyaluronan can be used to inhibit platelet activation, to inhibit
platelet binding to collagen, or to limit thrombosis or to form an
engineered collagen matrix. In any of these embodiments, any of the
above-described compounds can be used.
In another embodiment, a collagen-binding synthetic peptidoglycan
comprising a synthetic peptide of about 5 to about 40 amino acids
with amino acid sequence homology to a collagen binding peptide
(e.g. a portion of an amino acid sequence of a collagen binding
protein or proteoglycan) conjugated to heparin, dextran, or
hyaluronan can be used to inhibit platelet binding to collagen,
platelet activation, or both. In any of these embodiments, any of
the above-described compounds can be used.
In another embodiment, the synthetic peptides described herein can
be modified by the inclusion of one or more conservative amino acid
substitutions. As is well known to those skilled in the art,
altering any non-critical amino acid of a peptide by conservative
substitution should not significantly alter the activity of that
peptide because the side-chain of the replacement amino acid should
be able to form similar bonds and contacts as the side chain of the
amino acid which has been replaced.
Non-conservative substitutions are possible provided that these do
not excessively affect the collagen binding activity of the peptide
and/or reduce its effectiveness in altering the structure or
mechanical characteristics of a collagen matrix, in inhibiting
platelet activation, or in inhibiting platelet adhesion (e.g.
binding) to collagen.
As is well-known in the art, a "conservative substitution" of an
amino acid or a "conservative substitution variant" of a peptide
refers to an amino acid substitution which maintains: 1) the
secondary structure of the peptide; 2) the charge or hydrophobicity
of the amino acid; and 3) the bulkiness of the side chain or any
one or more of these characteristics. Illustratively, the
well-known terminologies "hydrophilic residues" relate to serine or
threonine. "Hydrophobic residues" refer to leucine, isoleucine,
phenylalanine, valine or alanine, or the like. "Positively charged
residues" relate to lysine, arginine, ornithine, or histidine.
"Negatively charged residues" refer to aspartic acid or glutamic
acid. Residues having "bulky side chains" refer to phenylalanine,
tryptophan or tyrosine, or the like. A list of illustrative
conservative amino acid substitutions is given in TABLE 1.
TABLE-US-00001 TABLE 1 For Amino Acid Replace With Alanine D-Ala,
Gly, Aib, .beta.-Ala, L-Cys, D-Cys Arginine D-Arg, Lys, D-Lys, Orn
D-Orn Asparagine D-Asn, Asp, D-Asp, Glu, D-Glu Gln, D-Gln Aspartic
Acid D-Asp, D-Asn, Asn, Glu, D-Glu, Gln, D-Gln Cysteine D-Cys,
S-Me-Cys, Met, D-Met, Thr, D-Thr Glutamine D-Gln, Asn, D-Asn, Glu,
D-Glu, Asp, D-Asp Glutamic Acid D-Glu, D-Asp, Asp, Asn, D-Asn, Gln,
D-Gln Glycine Ala, D-Ala, Pro, D-Pro, Aib, .beta.-Ala Isoleucine
D-Ile, Val, D-Val, Leu, D-Leu, Met, D-Met Leucine Val, D-Val, Met,
D-Met, D-Ile, D-Leu, Ile Lysine D-Lys, Arg, D-Arg, Orn, D-Orn
Methionine D-Met, S-Me-Cys, Ile, D-Ile, Leu, D-Leu, Val, D-Val
Phenylalanine D-Phe, Tyr, D-Tyr, His, D-His, Trp, D-Trp Proline
D-Pro Serine D-Ser, Thr, D-Thr, allo-Thr, L-Cys, D-Cys Threonine
D-Thr, Ser, D-Ser, allo-Thr, Met, D-Met, Val, D-Val Tyrosine D-Tyr,
Phe, D-Phe, His, D-His, Trp, D-Trp Valine D-Val, Leu, D-Leu, Ile,
D-Ile, Met, D-Met
In another embodiment, a collagen-binding synthetic peptidoglycan
comprising a synthetic peptide of about 5 to about 40 amino acids
with amino acid sequence homology to a portion of a collagen
binding peptide conjugated to heparin can be used to inhibit
platelet activation, inhibit platelet binding (e.g. adhesion) to
collagen, or to limit thrombosis or to form an engineered collagen
matrix. In another embodiment, the collagen-binding synthetic
peptidoglycan conjugated to dextran can be used to inhibit platelet
activation, inhibit platelet binding to collagen, or to limit
thrombosis or to form an engineered collagen matrix. In yet another
embodiment, the collagen-binding synthetic peptidoglycan conjugated
to hyaluronan can be used to inhibit platelet activation, inhibit
platelet binding to collagen, or to limit thrombosis or to form an
engineered collagen matrix. In any of these embodiments, any of the
above-described compounds can be used.
In another embodiment, a collagen-binding synthetic peptidoglycan
comprising a synthetic peptide of about 5 to about 40 amino acids
with amino acid sequence homology to a collagen binding peptide
(e.g. a portion of an amino acid sequence of a collagen binding
protein or a proteoglycan) conjugated to any glycan, such as, for
example, heparin, dextran, or hyaluronan can be used to inhibit
platelet binding to collagen, to inhibit platelet activation, or to
limit thrombosis. In any of these embodiments, any of the
above-described compounds can be used.
In one embodiment the synthetic peptide is synthesized according to
solid phase peptide synthesis protocols that are well known by
persons of skill in the art. In one embodiment a peptide precursor
is synthesized on a solid support according to the well-known Fmoc
protocol, cleaved from the support with trifluoroacetic acid and
purified by chromatography according to methods known to persons
skilled in the art.
In another embodiment the synthetic peptide is synthesized
utilizing the methods of biotechnology that are well known to
persons skilled in the art. In one embodiment a DNA sequence that
encodes the amino acid sequence information for the desired peptide
is ligated by recombinant DNA techniques known to persons skilled
in the art into an expression plasmid (for example, a plasmid that
incorporates an affinity tag for affinity purification of the
peptide), the plasmid is transfected into a host organism for
expression, and the peptide is then isolated from the host organism
or the growth medium according to methods known by persons skilled
in the art (e.g., by affinity purification). Recombinant DNA
technology methods are described in Sambrook et al., "Molecular
Cloning: A Laboratory Manual", 3rd Edition, Cold Spring Harbor
Laboratory Press, (2001), incorporated herein by reference, and are
well-known to the skilled artisan.
In one embodiment the synthetic peptide is conjugated to a glycan
by reacting a free amino group of the peptide with an aldehyde
function of the glycan in the presence of a reducing agent,
utilizing methods known to persons skilled in the art, to yield the
peptide glycan conjugate. In one embodiment an aldehyde function of
the glycan (e.g. polysaccharide or glycosaminoglycan) is formed by
reacting the glycan with sodium metaperiodate according to methods
known to persons skilled in the art.
In another embodiment the synthetic peptide is conjugated to a
glycan by reacting an aldehyde function of the glycan with
3-(2-pyridyldithio)propionyl hydrazide (PDPH) to form an
intermediate glycan and further reacting the intermediate glycan
with a peptide containing a free thiol group to yield the peptide
glycan conjugate. In yet another embodiment, the sequence of the
peptide may be modified to include a glycine-cysteine segment to
provide an attachment point for a glycan or a glycan-linker
conjugate.
Although specific embodiments have been described in the preceding
paragraphs, the collagen-binding synthetic peptidoglycans described
herein can be made by using any art-recognized method for
conjugation of the peptide to the glycan (e.g. polysaccharide or
glycosaminoglycan). This can include covalent, ionic, or hydrogen
bonding, either directly or indirectly via a linking group such as
a divalent linker. The conjugate is typically formed by covalent
bonding of the peptide to the glycan through the formation of
amide, ester or imino bonds between acid, aldehyde, hydroxy, amino,
or hydrazo groups on the respective components of the conjugate.
All of these methods are known in the art or are further described
in the Examples section of this application or in Hermanson G. T.,
Bioconjugate Techniques, Academic Press, pp. 169-186 (1996). The
linker typically comprises about 1 to about 30 carbon atoms, more
typically about 2 to about 20 carbon atoms. Lower molecular weight
linkers (i.e., those having an approximate molecular weight of
about 20 to about 500) are typically employed.
In addition, structural modifications of the linker portion of the
conjugates are contemplated herein. For example, amino acids may be
included in the linker and a number of amino acid substitutions may
be made to the linker portion of the conjugate, including but not
limited to naturally occurring amino acids, as well as those
available from conventional synthetic methods. In another aspect,
beta, gamma, and longer chain amino acids may be used in place of
one or more alpha amino acids. In another aspect, the linker may be
shortened or lengthened, either by changing the number of amino
acids included therein, or by including more or fewer beta, gamma,
or longer chain amino acids. Similarly, the length and shape of
other chemical fragments of the linkers described herein may be
modified.
In one aspect, the linker may include one or more bivalent
fragments selected independently in each instance from the group
consisting of alkylene, heteroalkylene, cycloalkylene,
cycloheteroalkylene, arylene, and heteroarylene each of which is
optionally substituted. As used herein heteroalkylene represents a
group resulting from the replacement of one or more carbon atoms in
a linear or branched alkylene group with an atom independently
selected in each instance from the group consisting of oxygen,
nitrogen, phosphorus and sulfur.
In one aspect, a collagen-binding synthetic peptidoglycan may be
administered to a patient (e.g., a patient in need of treatment to
inhibit platelet activation such as that involved in thrombosis).
In various embodiments, the collagen-binding synthetic
peptidoglycan can be administered intravenously, or into muscle, or
an internal organ, for example. Suitable routes for parenteral
administration include intravenous, intra-arterial, and
intramuscular delivery. Suitable means for parenteral
administration include needle (including microneedle) injectors and
infusion techniques.
In an illustrative embodiment, pharmaceutical formulations for use
with collagen-binding synthetic peptidoglycans for parenteral
administration comprising: a) a pharmaceutically active amount of
the collagen-binding synthetic peptidoglycan; b) a pharmaceutically
acceptable pH buffering agent to provide a pH in the range of about
pH 4.5 to about pH 9; c) an ionic strength modifying agent in the
concentration range of about 0 to about 300 millimolar; and d)
water soluble viscosity modifying agent in the concentration range
of about 0.25% to about 10% total formula weight or any
combinations of a), b), c) and d) are provided.
In various illustrative embodiments, the pH buffering agents for
use in the compositions and methods herein described are those
agents known to the skilled artisan and include, for example,
acetate, borate, carbonate, citrate, and phosphate buffers, as well
as hydrochloric acid, sodium hydroxide, magnesium oxide,
monopotassium phosphate, bicarbonate, ammonia, carbonic acid,
hydrochloric acid, sodium citrate, citric acid, acetic acid,
disodium hydrogen phosphate, borax, boric acid, sodium hydroxide,
diethyl barbituric acid, and proteins, as well as various
biological buffers, for example, TAPS, Bicine, Tris, Tricine,
HEPES, TES, MOPS, PIPES, cacodylate, or IVIES.
In another illustrative embodiment, the ionic strength modulating
agents include those agents known in the art, for example,
glycerin, propylene glycol, mannitol, glucose, dextrose, sorbitol,
sodium chloride, potassium chloride, and other electrolytes.
Useful viscosity modulating agents include but are not limited to,
ionic and non-ionic water soluble polymers; crosslinked acrylic
acid polymers such as the "carbomer" family of polymers, e.g.,
carboxypolyalkylenes that may be obtained commercially under the
Carbopol.RTM. trademark; hydrophilic polymers such as polyethylene
oxides, polyoxyethylene-polyoxypropylene copolymers, and
polyvinylalcohol; cellulosic polymers and cellulosic polymer
derivatives such as hydroxypropyl cellulose, hydroxyethyl
cellulose, hydroxypropyl methylcellulose, hydroxypropyl
methylcellulose phthalate, methyl cellulose, carboxymethyl
cellulose, and etherified cellulose; gums such as tragacanth and
xanthan gum; sodium alginate; gelatin, hyaluronic acid and salts
thereof, chitosans, gellans or any combination thereof. Typically,
non-acidic viscosity enhancing agents, such as a neutral or basic
agent are employed in order to facilitate achieving the desired pH
of the formulation.
In one illustrative aspect, parenteral formulations may be suitably
formulated as a sterile non-aqueous solution or as a dried form to
be used in conjunction with a suitable vehicle such as sterile,
pyrogen-free water. The preparation of parenteral formulations
under sterile conditions, for example, by lyophilisation, may
readily be accomplished using standard pharmaceutical techniques
well known to those skilled in the art.
In one embodiment, the solubility of a collagen-binding synthetic
peptidoglycan used in the preparation of a parenteral formulation
may be increased by the use of appropriate formulation techniques,
such as the incorporation of solubility-enhancing agents.
In various embodiments, formulations for parenteral administration
may be formulated to be for immediate and/or modified release.
Modified release formulations include delayed, sustained, pulsed,
controlled, targeted and programmed release formulations. Thus, a
collagen-binding synthetic peptidoglycan may be formulated as a
solid, semi-solid, or thixotropic liquid for administration as an
implanted depot providing modified release of the active compound.
Illustrative examples of such formulations include drug-coated
stents and copolymeric(dl-lactic, glycolic)acid (PGLA)
microspheres. In another embodiment, collagen-binding synthetic
peptidoglycans or compositions comprising collagen-binding
synthetic peptidoglycan may be continuously administered, where
appropriate.
In other embodiments, collagen-binding synthetic peptidoglycans and
compositions containing them can be administered topically. A
variety of dose forms and bases can be applied to the topical
preparations, such as an ointment, cream, gel, gel ointment,
plaster (e.g. cataplasm, poultice), solution, powders, and the
like. These preparations may be prepared by any conventional method
with conventional pharmaceutically acceptable carriers or diluents
as described below.
For example, in the preparation of an ointment, vaseline, higher
alcohols, beeswax, vegetable oils, polyethylene glycol, etc. can be
used. In the preparation of a cream formulation, fats and oils,
waxes, higher fatty acids, higher alcohols, fatty acid esters,
purified water, emulsifying agents etc. can be used. In the
preparation of gel formulations, conventional gelling materials
such as polyacrylates (e.g. sodium polyacrylate), hydroxypropyl
cellulose, hydroxypropyl methyl cellulose, polyvinyl alcohol,
polyvinylpyrrolidone, purified water, lower alcohols, polyhydric
alcohols, polyethylene glycol, and the like are used. In the
preparation of a gel ointment preparation, an emulsifying agent
(preferably nonionic surfactants), an oily substance (e.g. liquid
paraffin, triglycerides, and the like), etc. are used in addition
to the gelling materials as mentioned above. A plaster such as
cataplasm or poultice can be prepared by spreading a gel
preparation as mentioned above onto a support (e.g. fabrics,
non-woven fabrics). In addition to the above-mentioned ingredients,
paraffins, squalane, lanolin, cholesterol esters, higher fatty acid
esters, and the like may optionally be used. Moreover, antioxidants
such as BHA, BHT, propyl gallate, pyrogallol, tocopherol, etc. may
also be incorporated. In addition to the above-mentioned
preparations and components, there may optionally be used any other
conventional formulations for incorporated with any other
additives.
It is also contemplated that any of the formulations described
herein may be used to administer the collagen-binding synthetic
peptidoglycan (e.g., one or more types) either in the absence or
the presence of the engineered collagen matrices described
herein.
In various embodiments, the dosage of the collagen-binding
synthetic peptidoglycan, with or without an engineered collagen
matrix, can vary significantly depending on the patient condition,
the disease state being treated, the route of administration and
tissue distribution, and the possibility of co-usage of other
therapeutic treatments. The effective amount to be administered to
a patient is based on body surface area, patient weight or mass,
and physician assessment of patient condition. In various exemplary
embodiments, an effective dose can range from about 1 ng/kg to
about 10 mg/kg, 100 ng/kg to about 1 mg/kg, from about 1 .mu.g/kg
to about 500 .mu.g/kg, or from about 100 .mu.g/kg to about 400
.mu.g/kg. In each of these embodiments, dose/kg refers to the dose
per kilogram of patient mass or body weight. In other illustrative
aspects, effective doses can range from about 0.01 .mu.g to about
1000 mg per dose, 1 .mu.g to about 100 mg per dose, or from about
100 .mu.g to about 50 mg per dose, or from about 500 .mu.g to about
10 mg per dose or from about 1 mg to 10 mg per dose.
Any effective regimen for administering the collagen-binding
synthetic peptidoglycan can be used. For example, the
collagen-binding synthetic peptidoglycan can be administered as a
single dose, or as a multiple-dose daily regimen. Further, a
staggered regimen, for example, one to five days per week can be
used as an alternative to daily treatment.
In one embodiment of the invention the patient is treated with
multiple injections of the collagen-binding synthetic
peptidoglycan. In one embodiment, the patient is injected multiple
times (e.g., about 2 up to about 50 times) with the
collagen-binding synthetic peptidoglycan, for example, at 12-72
hour intervals or at 48-72 hour intervals. Additional injections of
the collagen-binding synthetic peptidoglycan can be administered to
the patient at an interval of days or months after the initial
injections(s) and the additional injections prevent recurrence of
disease. Alternatively, the initial injection(s) of the
collagen-binding synthetic peptidoglycan may prevent recurrence of
disease.
In any of the embodiments herein described, it is to be understood
that a combination of two or more collagen-binding synthetic
peptidoglycans, differing in the peptide portion, the glycan
portion, or both, can be used in place of a single collagen-binding
synthetic peptidoglycan.
It is also appreciated that in the foregoing embodiments, certain
aspects of the compounds, compositions and methods are presented in
the alternative in lists, such as, illustratively, selections for
any one or more of G and P. It is therefore to be understood that
various alternate embodiments of the invention include individual
members of those lists, as well as the various subsets of those
lists. Each of those combinations are to be understood to be
described herein by way of the lists.
In the following illustrative examples, the terms "synthetic
peptidoglycan" and "conjugate" are used synonymously with the term
"collagen-binding synthetic peptidoglycan."
EXAMPLE 1
Peptide Synthesis
All peptides were synthesized using a Symphony peptide synthesizer
(Protein Technologies, Tucson, Ariz.), utilizing an FMOC protocol
on a Knorr resin. The crude peptide was released from the resin
with TFA and purified by reverse phase chromatography on an
AKTAexplorer (GE Healthcare, Piscataway, NJ) utilizing a
Grace-Vydac 218TP C-18 reverse phase column and a gradient of
water/acetonitrile 0.1% TFA. Dansyl-modified peptides were prepared
by adding an additional coupling step with dansyl-Gly (Sigma)
before release from the resin. Peptide structures were confirmed by
mass spectrometry. The following peptides were prepared as
described above: RRANAALKAGELYKSILYGC [SEQ ID NO: 1], SYIRIADTNIT
[SEQ ID NO: 10], Dansyl-GRRANAALKAGELYKSILYGC [SEQ ID NO: 15], and
Dansyl-GSYIRIADTNIT [SEQ ID NO: 16]. These peptides are abbreviated
SILY, SYIR, Z-SILY, and Z-SYIR. Additional peptides, KELNLVYTGC
[SEQ ID NO: 12] (abbreviated KELN) and GSITTIDVPWNVGC [SEQ ID NO:
14] (abbreviated GSIT) were prepared as described above or
purchased (Genescript, Piscataway, NJ).
EXAMPLE 2
Conjugation of SYIR Peptide to Dermatan Sulfate
SYIR was conjugated to oxDS by a method adapted from Hermanson with
slight modifications (Hermanson, 1996). The peptide SYIR was
dissolved in 0.05M sodium carbonate, 0.1M sodium citrate buffer, pH
9.5, at a concentration of 0.4 mg/mL for a final volume of 5 mL. To
react in 10-fold peptide molar excess, 29 mg of oxDS MW 41,000
(oxidized dermatan sulfate, containing 1.1 aldehydes/DS molecule of
41 kDa is available from Celsus Laboratories, Cincinnati, Ohio) was
dissolved into the peptide solution. Under gentle stirring, 50
.mu.L sodium cyanoborohydride was added, and the reaction allowed
to proceed at room temperature overnight.
Excess peptide was separated by gel filtration on an Akta Purifier
using an XK 26-40 column packed with Sephadex G-25 medium (GE
Health) and equilibrated with deionized water (MilliQ). Eluent was
monitored at 215 nm, 254 nm, and 280 nm. The first eluting peak
containing DS-SYIR was collected and lyophilized for further
testing.
EXAMPLE 3
Conjugation of SILY to Dermatan Sulfate
PDPH Attachment to oxDS
The bifunctional crosslinker PDPH (Pierce), reactive to sulfhydryl
and amine groups, was used to conjugate SILY to oxDS. In the first
step of the reaction, oxDS was dissolved in coupling buffer (0.1M
sodium phosphate, 0.25M sodium chloride, pH 7.2) to a final
concentration of 1.2 mM. PDPH was added in 10-fold molar excess,
and the reaction proceeded at room temperature for 2 hours. Excess
PDPH (MW 229Da) was separated by gel filtration on an Akta Purifier
using an XK 26-40 column packed with Sephadex G-25 medium and
equilibrated with MilliQ water. Eluent was monitored at 215 nm, 254
nm, and 280 nm. The first eluting peak containing DS-PDPH was
collected and lyophilized for conjugating with SILY.
Determination of PDPH Content
To determine the number of PDPH molecules conjugated to oxDS.
DS-PDPH was dissolved in coupling buffer at 1.6 mg/mL. 10 .mu.L of
DTT at 15 mg/mL was added to the DS-PDPH solution, and the reaction
proceeded at room temperature for 15 min. Reducing the disulfide
bond on the cysteine reactive side of PDPH liberates
pyridine-2-thione, which is visible at 313 nm. Absorbance at 313 nm
was measured before and after the addition of DTT, and the
difference was used to calculate the number of PDPH molecules/DS
molecule using the extinction coefficient of pyridine-2-thione.
Results in FIG. 7. show .DELTA.A=0.35, corresponding to 1.1 PDPH
molecules/DS.
Conjugation of SILY
The peptide was dissolved in a 5:1 molar excess in coupling buffer
at a final peptide concentration of approximately 1mM (limited by
peptide solubility). The reaction was allowed to proceed at room
temperature overnight, and excess peptide was separated and the
DS-SILY conjugate isolated by gel filtration as described above.
See FIG. 26 showing a SILY/DS ratio of 1.06 after coupling.
EXAMPLE 4
Conjugation of Z-SILY to Dermatan Sulfate
Dermatan sulfate was conjugated to Z-SILY according to the method
of EXAMPLE 3.
EXAMPLE 5
Conjugation of KELN to Dermatan Sulfate
Dermatan sulfate was conjugated to KELN according to the method of
EXAMPLE 3.
EXAMPLE 6
Conjugation of GSIT to Dermatan Sulfate
Dermatan sulfate was conjugated to GSIT according to the method of
EXAMPLE 3.
EXAMPLE 7
Conjugation of Z-SYIR to Dermatan Sulfate
Dermatan sulfate was conjugated to Z-SYIR according to the method
of EXAMPLE 2.
EXAMPLE 8
Conjugation of SILY to Heparin
Oxidized Heparin (oxHep) (MW=19.7 kDa) containing 1 aldehyde per
molecule (purchased from Celsus Laboratories, Cincinnati, OH).
Additional aldehydes were formed by further oxidation in sodium
meta-periodate as follows. oxHep was dissolved in 0.1M sodium
acetate pH 5.5 at a concentration of 10 mg/mL. Sodium
meta-periodate was then added at a concentration of 2 mg/mL and
allowed to react for 4 hours at room temperature protected from
light. Excess sodium meta-periodate was removed by desalting using
a HiTrap size exclusion column (GE Healthcare) and oxHep was
lyophilized protected from light until conjugation with PDPH
(3-(2-pyridyldithio)propionyl hydrazide).
oxHep was conjugated to PDPH (3-(2-pyridyldithio)propionyl
hydrazide)by the method described for DS-PDPH conjugation, EXAMPLE
3. PDPH was reacted in 50-fold molar excess. To achieve a higher
PDPH concentration, 10 mg PDPH was dissolved in 75 .mu.L DMSO and
mixed with 1 mL coupling buffer containing oxHep. The reaction
proceeded at room temperature for 2.5 hours and excess PDPH was
removed by desalting. Heparin containing PDPH (Hep-PDPH) was stored
as a lyophilized powder until reacted with SILY.
SILY was reacted in 10-fold molar excess with Hep-PDPH as described
for DS-SILY conjugation in EXAMPLE 3. The reaction was monitored as
described for DS-SILY in EXAMPLE 3 and showed 5.44 SILY peptides
conjugated per heparin molecule as shown in FIG. 24.
EXAMPLE 9
Conjugation of GSIT to Heparin
Heparin was conjugated to GSIT according to the method of EXAMPLE 8
(abbreviated Hep-GSIT).
EXAMPLE 10
Conjugation of SILY to Dextran
Dextran was conjugated to SILY according to the method of EXAMPLE 8
replacing heparin with dextran. Modification of the conditions for
oxidation of dextran with sodium meta-periodate in the first step
to allowed preparation of conjugates with different molar ratios of
SILY to dextran. For example dextran-SILY conjugates with a molar
ratio of SILY to dextran of about 6 and a dextran-SILY conjugate
with a molar ratio of SILY to dextran of about 9 were prepared
(abbreviated Dex-SILY6 and Dex-SILY9).
EXAMPLE 11
Conjugation of SILY to Hyaluronan
Hyaluronan was conjugated to SILY according to the method of
EXAMPLE 8 (abbreviated HA-SILY).
EXAMPLE 12
SILY Binding to Collagen (Biacore)
Biacore studies were performed on a Biacore 2000 using a CM-3 chip
(Biacore, Inc., Piscataway, NJ). The CM-3 chip is coated with
covalently attached carboxymethylated dextran, which allows for
attachment of the substrate collagen via free amine groups. Flow
cells (FCs) 1 and 2 were used, with FC-1 as the reference cell and
FC-2 as the collagen immobilized cell. Each FC was activated with
EDC-NHS, and 1500 RU of collagen was immobilized on FC-2 by flowing
1 mg/mL collagen in sodium acetate, pH 4, buffer at 5 .mu.L/min for
10 min. Unreacted NHS-ester sites were capped with ethanolamine;
the control FC-1 was activated and capped with ethanolamin.
To determine peptide binding affinity, SILY was dissolved in
1.times.HBS-EP buffer (Biacore) at varying concentrations from 100
uM to 1.5 .mu.m in 2-fold dilutions. The flow rate was held at 90
.mu.L/min which is in the range suggested by Myska for determining
binding kinetics (Myska, 1997). The first 10 injections were buffer
injections, which help to prime the system, followed by randomized
sample injections, run in triplicate. Analysis was performed using
BIAevaluation software (Biacore). Representative
association/disassociation curves are shown in FIG. 3 demonstrating
that the SILY peptide binds reversibly with collagen. K.sub.D=1.2
.mu.M was calculated from the on-off binding kinetics.
EXAMPLE 13
Z-SILY Binding to Collagen
Binding assays were done in a 96-well high-binding plate, black
with a clear bottom (Costar). Collagen was compared to untreated
wells and BSA coated wells. Collagen and BSA were immobilized at
37.degree. C. for 1 hr by incubating 90 .mu.L/well at
concentrations of 2 mg/mL in 10 mM HCl and 1.times.PBS,
respectively. Each well was washed 3.times. with 1.times.PBS after
incubating. Z-SILY was dissolved in 1.times.PBS at concentrations
from 100 .mu.M to 10 nM in 10-fold dilutions. Wells were incubated
for 30 min at 37.degree. C. and rinsed 3.times. with PBS and then
filled with 90 .mu.L of 1.times.PBS. Fluorescence readings were
taken on an M5 Spectramax Spectrophotometer (Molecular Devices) at
excitation/emission wavelengths of 335 nm/490 nm respectively. The
results are shown in FIGS. 4 and 5. K.sub.D=0.86 .mu.M was
calculated from the equilibrium kinetics.
EXAMPLE 14
Characterizing DS-SILY
To determine the number of SILY molecules conjugated to DS, the
production of pyridine-2-thione was measured using a modified
protocol provided by Pierce. Dermatan sulfate with 1.1 PDPH
molecules attached was dissolved in coupling buffer (0.1M sodium
phosphate, 0.25M sodium chloride) at a concentration of 0.44 mg/mL
and absorbance at 343 nm was measured using a SpectraMax M5
(Molecular Devices). SILY was reacted in 5-fold molar excess and
absorbance measurements were repeated immediately after addition of
SILY and after allowing to react for 2 hours. To be sure SILY does
not itself absorb at 343 nm, coupling buffer containing 0.15 mg/mL
SILY was measured and was compared to absorbance of buffer
alone.
The number of SILY molecules conjugated to DS was calculated by the
extinction coefficient of pyridine-2-thione using the following
equation (Abs.sub.343/8080).times.(MW.sub.DS/DS.sub.mg/mL). The
results are shown in FIG. 26.
EXAMPLE 15
Collagen Binding, Fluorescence Data--DS-SILY
In order to determine whether the peptide conjugate maintained its
ability to bind to collagen after its conjugation to DS, a
fluorescent binding assay was performed. A fluorescently labeled
version of SILY, Z-SILY, was synthesized by adding dansylglycine to
the amine terminus. This peptide was conjugated to DS and purified
using the same methods described for SILY.
Binding assays were done in a 96-well high binding plate, black
with a clear bottom (Costar). Collagen was compared to untreated
wells and BSA coated wells. Collagen and BSA were immobilized at
37.degree. C. for 1 hr by incubating 90 .mu.L/well at
concentrations of 2 mg/mL in 10 mM HCl and 1.times.PBS
respectively. Each well was washed 3.times. with 1.times.PBS after
incubating.
Wells were preincubated with DS at 37.degree. C. for 30 min to
eliminate nonspecific binding of DS to collagen. Wells were rinsed
3.times. with 1.times.PBS before incubating with DS-Z-SILY.
DS-Z-SILY was dissolved in 1.times.PBS at concentrations from 100
.mu.M to 10 nM in 10-fold dilutions. Wells were incubated for 30
min at 37.degree. C. and rinsed 3.times. and then filled with 90
.mu.L of 1.times.PBS. Fluorescence readings were taken on an M5
Spectramax Spectrophotometer (Molecular Devices) at
excitation/emission wavelengths of 335 nm/490 nm respectively.
Fluorescence binding of DS-Z-SILY on immobilized collagen, BSA, and
untreated wells are compared in FIG. 8. Results show that DS-Z-SILY
binds specifically to the collagen-treated wells over BSA and
untreated wells. The untreated wells of the high bind plate were
designed to be a positive control, though little binding was
observed relative to collagen treated wells. These results suggest
that SILY maintains its ability to bind to collagen after it is
conjugated to DS. Preincubating with DS did not prevent binding,
suggesting that the conjugate binds separately from DS alone.
EXAMPLE 16
Preparation of Type I Collagen Gels
Gels were made with Nutragen collagen (Inamed, Freemont, Calif.) at
a final concentration of 4 mg/mL collagen. Nutragen stock is 6.4
mg/mL in 10 mM HCl. Gel preparation was performed on ice, and fresh
samples were made before each test. The collagen solution was
adjusted to physiologic pH and salt concentration, by adding
appropriate volumes of 10.times.PBS (phosphate buffered saline),
1.times.PBS, and 1M NaOH. For most experiments, samples of DS,
decorin, DS-SILY, or DS-SYIR were added at a 10:1 collagen:sample
molar ratio by a final 1.times.PBS addition (equal volumes across
treatments) in which the test samples were dissolved at appropriate
concentrations. In this way, samples are constantly kept at pH 7.4
and physiologic salt concentration. Collagen-alone samples received
a 1.times.PBS addition with no sample dissolved. Fibrillogenesis
will be induced by incubating neutralized collagen solutions at
37.degree. C. overnight in a humidified chamber to avoid
dehydration. Gel solutions with collagen:sample molar ratios of
other than 10:1 were prepared similarly.
EXAMPLE 17
Viscoelastic Characterization of Gels
Collagen gels were prepared as described in EXAMPLE 16 and prior to
heating, 200 .mu.L of each treatment were pipetted onto the
wettable surface of hydrophobically printed slides (Tekdon). The
PTFE printing restricted gels to the 20 mm diameter wettable
region. Gels were formed in a humidified incubator at 37.degree. C.
overnight prior to mechanical testing.
Slides were clamped on the rheometer stage of a AR-G2 rheometer
with 20 mm stainless steel parallel plate geometry (TA Instruments,
New Castle, Del.), and the 20 mm stainless steel parallel plate
geometry was lowered to a gap distance of 600 .mu.m using a normal
force control of 0.25N to avoid excessive shearing on the formed
gel. An iterative process of stress and frequency sweeps was
performed on gels of collagen alone to determine the linear range.
All samples were also tested over a frequency range from 0.1 Hz to
1.0 Hz and a controlled stress of 1.0 Pa. Statistical analysis
using Design Expert software (StatEase, Minneapolis, Minn.) was
performed at each frequency and a 5-way ANOVA used to compare
samples. The results shown in FIG. 9, 10:1; FIG. 10, 5:1; and FIG.
11, 30:1 demonstrate that treatment with synthetic peptidoglycans
can modify the viscoelastic behavior of collagen type I gels.
EXAMPLE 18
Viscoelastic Characterization of Collagen III Containing Gels
Gels containing type III collagen were prepared as in EXAMPLE 16
with the following modifications: treated and untreated gel
solutions were prepared using a collagen concentration of 1.5 mg/mL
(90% collagen III (Millipore), 10% collagen I), 200 .mu.L samples
were pipetted onto 20 mm diameter wettable surfaces of hydrophobic
printed slides. These solutions were allowed to gel at 37.degree.
C. for 24 hours. Gels were formed from collagen alone, collagen
treated with dermatan sulfate (1:1 and 5:1 molar ratio), and
collagen treated with the collagen III-binding peptides alone (GSIT
and KELN, 5:1 molar ratio) served as controls. The treated gels
contained the peptidoglycans (DS-GSIT or DS-KELN at 1:1 and 5:1
molar ratios. All ratios are collagen:treatment compound ratios.
The gels were characterized as in EXAMPLE 17, except the samples
were tested over a frequency range from 0.1 Hz to 1.0 Hz at a
controlled stress of 1.0 Pa. As shown in FIGS. 12 and 13, the
dermatan sulfate-GSIT conjugate and the dermatan sulfate-KELN
conjugate (synthetic peptidoglycans) can influence the viscoelastic
properties of gels formed with collagen type III.
EXAMPLE 19
Fibrillogenesis
Collagen fibrillogenesis was monitored by measuring turbidity
related absorbance at 313 nm providing information on rate of
fibrillogenesis and fibril diameter. Gel solutions were prepared as
described in EXAMPLE 16 (4 mg/mL collagen, 10:1 collagen:treatment,
unless otherwise indicated) and 50 uL/well were added at 4.degree.
C. to a 384-well plate. The plate was kept at 4.degree. C. for 4
hours before initiating fibril formation. A SpectraMax M5 at
37.degree. C. was used to measure absorbance at 313 nm at 30 s
intervals for 6 hours. The results are shown in FIGS. 14, 15, and
16. The T.sub.1/2 for gel formation of the 10:1 molar ratio samples
is shown in FIG. 17. Dermatan sulfate-SILY decreases the rate of
fibrillogenesis.
EXAMPLE 20
Confocal Reflection Microscopy
Gels were formed and incubated overnight as described above in
EXAMPLE 16, the gels were imaged with an Olympus FV1000 confocal
microscope using a 60.times., 1.4 NA water immersion lens. Samples
were illuminated with 488 nm laser light and the reflected light
was detected with a photomultiplier tube using a blue reflection
filter. Each gel was imaged 100 .mu.M from the bottom of the gel,
and three separate locations were imaged to ensure representative
sampling. Results are shown in FIG. 18.
EXAMPLE 21
Cryo-SEM Measurements on Collagen I
Gels for cryo-SEM were formed, as in EXAMPLE 16, directly on the
SEM stage and incubated at 37.degree. C. overnight. The stages were
then secured in a cryo-holder and plunged into liquid nitrogen
slush. Samples were then transferred to a Gatan Alto 2500
pre-chamber cooled to -170.degree. C. under vacuum. A free-break
surface was created with a cooled scalpel, and each sample
evaporated under sublimation conditions for 20 min. The sample was
coated by platinum sputter coating for 120 s. Samples were
transferred to the cryo-stage at -130.degree. C. and regions with
similar orientation were imaged for comparison across treatments.
Representative samples imaged at 5,000.times. are shown in FIG. 19.
Analysis of the images was performed to determine the average
fibril diameter, FIG. 22; and the average distance between collagen
sheets, FIG. 23. Fibril diameter was calculated using ImageJ
software (NIH) measuring individual fibrils by hand (drawing a line
across fibrils and measuring its length after properly setting the
scale). There were 3 observers, 3 separate images per treatment, 10
fibrils recorded per image giving a total of 90 measurements per
treatment. Sheet distance was calculated using ImageJ, again
measuring by hand. One observer and 15 measurements per treatment.
Fibril diameter and distance between collagen sheets decreased in
the gels treated with the dermatan sulfate-SILY synthetic
peptidoglycan.
EXAMPLE 22
Cryo-SEM Measurements on Collagen III
Gels for cryo-SEM were formed, as in EXAMPLE 16, directly on the
SEM stage and incubated at 37.degree. C. overnight with the
following modifications. The collagen concentration was 1 mg/mL
(90% collagen III, 10% collagen I). The collagen:DS ratio was 1:1
and the collagen:peptidoglycan ratio was 1:1. The images were
recorded as in EXAMPLE 21. The ratio of void volume to fibril
volume was measured using a variation of the method in EXAMPLE 21.
The results are shown in FIGS. 20 and 21. Dermatan sulfate-KELN and
dermatan sulfate-GSIT decrease void space (increase fibril diameter
and branching) in the treated collagen gels.
EXAMPLE 23
AFM Confirmation of D-Banding
Gel solutions were prepared as described in EXAMPLE 16 and 20 .mu.L
of each sample were pipetted onto a glass coverslip and allowed to
gel overnight in a humidified incubator. Gels were dehydrated by
treatment with graded ethanol solutions (35%, 70%, 85%, 95%, 100%),
10 min in each solution. AFM images were made in contact mode, with
a scan rate of 2 Hz (Multimode SPM, Veeco Instruments, Santa
Barbara, Calif., USA, AFM tips Silicon Nitride contact mode tip
k=0.05N/m, Veeco Instruments) Deflection setpoint: 0-1 Volts.
D-banding was confirmed in all treatments as shown in FIGS. 2 and
38.
EXAMPLE 24
Collagen Remodeling
Tissue Sample Preparation
Following a method by Grassl, et al. (Grassl, et al., Journal of
Biomedical Materials Research 2002, 60, (4), 607-612), which is
herein incorporated in its entirety, collagen gels with or without
synthetic PG mimics were formed as described in EXAMPLE 16. Human
aortic smooth muscle cells (Cascade Biologics, Portland, Oreg.)
were seeded within collagen gels by adding 4.times.10.sup.6
cells/mL to the neutralized collagen solution prior to incubation.
The cell-collagen solutions were pipetted into an 8-well Lab-Tek
chamber slide and incubated in a humidified 37.degree. C. and 5%
CO.sub.2 incubator. After gelation, the cell-collagen gels will be
covered with 1 mL Medium 231 as prescribed by Cascade. Every 3-4
days, the medium was removed from the samples and the
hydroxyproline content measured by a standard hydroxyproline assay
(Reddy, 1996).
Hydroxyproline Content
To measure degraded collagen in the supernatant medium, the sample
was lyophilized, the sample hydrolyzed in 2M NaOH at 120.degree. C.
for 20 min. After cooling, free hydroxyproline was oxidized by
adding chloramine-T (Sigma) and reacting for 25 min at room
temperature. Ehrlich's aldehyde reagent (Sigma) was added and
allowed to react for 20 min at 65.degree. C. and followed by
reading the absorbance at 550 nm on an M-5 spectrophotometer
(Molecular Devices). Hydroxyproline content in the medium is an
indirect measure degraded collagen and tissue remodeling potential.
Cultures were incubated for up to 30 days and three samples of each
treatment measured. A gels incubated without added cells were used
as a control. Free peptides SILY and Dc13 resulted in greater
collagen degradation compared to collagen alone as measured by
hydroxyproline content in cell medium as shown in FIG. 53.
Cell Viability
Cell viability was determined using a live/dead violet
viability/vitality kit (Molecular Probes. The kit contains
calcein-violet stain (live cells) and aqua-fluorescent reactive dye
(dead cells). Samples were washed with 1.times.PBS and incubated
with 300 .mu.L of dye solution for 1 hr at room temperature. To
remove unbound dye, samples were rinsed with 1.times.PBS. Live and
dead cells were counted after imaging a 2-D slice with filters
400/452 and 367/526 on an Olympus FV1000 confocal microscope with a
20.times. objective. Gels were scanned for representative regions
and 3 image sets were taken at equal distances into the gel for all
samples.
EXAMPLE 25
Cell Proliferation in Gels
Gel samples were prepared as in EXAMPLE 16 (4 mg/mL collagen, 10:1
collagen:treatment) Cells were seeded at 1.5.times.10.sub.4
cells/cm.sup.2 and were incubated in growth medium for 4 hrs to
adhere the cells to the gel. The growth medium was then aspirated
and the cells were treated for 24 hrs. Treatment concentrations
were equal to those in gels at 10:1 molar ratio collagen:
treatment. The cells were incubated in growth medium for 4 hrs to
adhere to the gel. The growth medium was removed by aspiration and
replaced with fresh growth medium. The samples were incubated for
24 h. The number of cells in each sample was measured using the
CyQuant Cell Proliferation Assay (Invitrogen, Carlsbad, Calif.,
USA). The results shown in FIG. 25 indicate that the synthetic
peptidoglycans and peptides do not adversely affect cell
proliferation.
EXAMPLE 26
Preparation of DS-Dc13
The Dc13 peptide sequence is SYIRIADTNITGC and its fluorescently
labeled form is ZSYIRIADTNITGC, where Z designates dansylglycine.
Conjugation to dermatan sulfate using the heterobifunctional
crosslinker PDPH is performed as described for DS-SILY in EXAMPLE
3. As shown in FIG. 27, the molar ratio of Dc13 to dermatan sulfate
in the conjugate (DS-Dc13) was about 1.
EXAMPLE 27
Fluorescence Binding Assay For DS-ZSILY
The fluorescence binding assays described for DS-ZSILY was
performed with peptide sequence ZSYIRIADTNITGC (ZDc13). The results
appear in FIG. 28, showing that DS-ZDc13 binds specifically to the
collagen surface in a dose-dependent manner, though saturation was
not achieved at the highest rate tested.
EXAMPLE 28
Fibrillogenesis Assay For DS-Dc13
A fibrillogenesis assay as described for DS-SILY, EXAMPLE 19,
performed with the conjugate DS-Dc13. The results shown in FIG. 29
indicate that the DS-Dc13 delays fibrillogenesis and decreases
overall absorbance in a dose-dependent manner. Free Dc13 peptide in
contrast has little effect on fibrillogenesis compared to collagen
alone at the high 1:1 collagen:additive molar ratio.
EXAMPLE 29
Use of Cryo-SEM to Measure Fibril Diameters
Using a modification of EXAMPLE 21 fibril diameters were measured
by cryo-SEM. Fibril diameters from cryo-SEM images taken at
20,000.times. were measured using ImageJ software (NIH). At least
45 fibrils were measured for each treatment. Results are presented
as Avg..+-.S.E. Statistical analysis was performed using
DesignExpert software (StatEase) with .alpha.=0.05. The results are
shown in FIG. 30. Decorin and synthetic peptidoglycans
significantly decrease fibril diameter over collagen or
collagen+dermatan sulfate. Compared to collagen alone, free peptide
Dc13 does not affect fibril diameter while free SILY results in a
decrease in fibril diameter.
EXAMPLE 30
Cell Culture and Gel Compaction
Human coronary artery smooth muscle cells (HCA SMC) (Cascade
Biologics) were cultured in growth medium (Medium 231 supplemented
with smooth muscle growth factor). Cells from passage 3 were used
for all experiments. Differentiation medium (Medium 231
supplemented with 1% FBS and 1.times. pen/strep) was used for all
experiments unless otherwise noted. This medium differs from
manufacturer protocol in that it does not contain heparin.
Collagen gels were prepared with each additive as described with
the exception that the 1.times.PBS example addition was omitted to
accommodate the addition of cells in media. After incubating on ice
for 30 min, HCA SMCs in differentiation medium were added to the
gel solutions to a final concentration of 1.times.10.sup.6
cells/mL. Gels were formed in quadruplicate in 48-well non-tissue
culture treated plates (Costar) for 6 hrs before adding 500
.mu.L/well differentiation medium. Gels were freed from the well
edges after 24 hrs. Medium was changed every 2-3 days and images
for compaction were taken at the same time points using a Gel Doc
System (Bio-Rad). The cross-sectional area of circular gels
correlating to degree of compaction was determined using ImageJ
software (NIH). Gels containing no cells were used as a negative
control and cells in collagen gels absent additive were used as a
positive control. The results are shown in FIG. 31. By day 10 all
gels had compacted to approximately 10% of the original gel area,
and differences between additives were small. Gels treated with
DS-Dc13 were slightly, but significantly, less compact than gels
treated with decorin or collagen but compaction was statistically
equivalent to that seen with DS and DS-SILY treated gels.
EXAMPLE 31
Measurement of Elastin
Collagen gels seeded with HCA SMCs were prepared as described in
EXAMPLE 30. Differentiation medium was changed every three days and
gels were cultured for 10 days. Collagen gels containing no cells
were used as a control. Gels were rinsed in 1.times.PBS overnight
to remove serum protein, and gels were tested for elastin content
using the Fastin elastin assay per manufacturers protocol
(Biocolor, County Atrim, U.K.). Briefly, gels were solubilized in
0.25 M oxalic acid by incubating at 100.degree. C. for 1 hr.
Elastin was precipitated and samples were then centrifuged at
11,000.times.g for 10 min. The solubilized collagen supernatant was
removed and the elastin pellet was stained by Fastin Dye Reagent
for 90 min at room temperature. Samples were centrifuged at
11,000.times.g for 10 min and unbound dye in the supernatant was
removed. Dye from the elastin pellets was released by the Fastin
Dye Dissociation Reagent, and 100 .mu.L samples were transferred to
a 96-well plate (Costar). Absorbance was measured at 513 nm, and
elastin content was calculated from an .alpha.-elastin standard
curve. The results of these assays are shown in FIG. 32. Treatment
with DS-SILY significantly increased elastin production over all
samples. Treatment with DS and DS-Dc13 significantly decreased
elastin production over untreated collagen. Control samples of
collagen gels with no cells showed no elastin production.
EXAMPLE 32
Effect of Heparin or Heparin-SILY on Platelet Interaction
Collagen was immobilized on glass cover slides (18 mm) by
incubating slides with collagen at 2 mg/mL in 10 mM HCl for 1 hr at
37.degree. C. Slides were then washed with 1.times.PBS and stored
at 4.degree. C. in 1.times.PBS for 24 hrs until further testing.
Untreated glass cover slides were used as a negative control.
Slides were placed into a 48-well non tissue-culture treated plate
(Costar) with the collagen surface facing up. Heparin or
Heparin-SILY were dissolved in 1.times.PBS to a concentration of
100 .mu.M and incubated at 100 .mu.L/well for 30 min at 37.degree.
C. Unbound heparin or Heparin-SILY were aspirated and the surfaces
were washed with 1 mL 1.times.PBS. Collagen immobilized slides
incubated with 1.times.PBS containing no additive were used as a
positive control.
Whole human blood was centrifuged at 800.times.g for 15 min and 100
.mu.L of platelet-rich plasma was removed from the buffy coat layer
and added to each well. After incubating for 1 hr at 37.degree. C.,
platelet-rich plasma was removed from the wells and the wells were
gently washed with 1.times.PBS to remove unbound cells. Slides were
fixed with 5% glutaraldehyde for 1 hr at room temperature, rinsed,
and lyophilized before imaging. Slides were gold sputter coated for
3 min and imaged at 200.times. on a JEOL 840 SEM. The results are
shown in FIG. 33. This images show that treatment with the
heparin-SILY conjugate affects platelet cell binding to
collagen.
EXAMPLE 33
Cryo-SEM Measurement of Fibril Density
Collagen gels were formed in the presence of each additive at a
10:1 molar ratio, as described in EXAMPLE 16, directly on the SEM
stage, processed, and imaged as described. Images at 10,000.times.
were analyzed for fibril density calculations. Images were
converted to 8-bit black and white, and threshold values for each
image were determined using ImageJ software (NIH). The threshold
was defined as the value where all visible fibrils are white, and
all void space is black. The ratio of white to black area was
calculated using MatLab software. All measurements were taken in
triplicate and thresholds were determined by an observer blinded to
the treatment. Images of the gels are shown in FIG. 37 and the
measured densities are shown in FIG. 34.
EXAMPLE 34
Viscoelastic Characterization of Gels Containing Dc13 or
DS-Dc13
Collagen gels were prepared, as in EXAMPLE 16. Viscoelastic
characterization was performed as described in EXAMPLE 17 on gels
formed with varying ratios of collagen to additive (treatment).
Treatment with dermatan sulfate or dermatan-Dc13 conjugate increase
the stiffness of the resulting collagen gel over untreated collagen
as shown in FIG. 35.
EXAMPLE 35
Cell Proliferation and Cytotoxicity Assay
HCA SMCs, prepared as in EXAMPLE 30, were seeded at
4.8.times.10.sup.4 cells/mL in growth medium onto a 96-well
tissue-culture black/clear bottom plate (Costar) and allowed to
adhere for 4 hrs. Growth medium was aspirated and 600 .mu.L of
differentiation medium containing each additive at a concentration
equivalent to the concentration within collagen gels
(1.4.times.10.sup.-6 M) was added to each well. Cells were
incubated for 48 hrs and were then tested for cytotoxicity and
proliferation using Live-Dead and CyQuant (Invitrogen) assays,
respectively, according to the manufacturer's protocol. Cells in
differentiation medium containing no additive were used as control.
The results are shown in FIG. 36 indicating that none of the
treatments demonstrated significant cytotoxic effects.
EXAMPLE 36
Inhibition of Platelet Binding and Platelet Activation to Collagen
Type I
Microplate Preparation
Type I fibrillar collagen (Chronolog, Havertown, PA) was diluted in
isotonic glucose to a concentration of 20-100 .mu.g/mL. 50 .mu.L of
collagen solution was added to each well of a high bind 96-well
plate. The plate was incubated overnight at 4 C, and then rinsed
3.times. with 1.times.PBS.
Peptidoglycan was diluted in 1.times.PBS at concentrations of 25
.mu.M to 50 .mu.M and 50 .mu.L solution was added to the collagen
coated wells. Controls of GAG, peptide, or PBS were also added to
collagen coated wells as controls. Treatments were incubated at
37.degree. C. with shaking at 200 rpm for 30 min. Wells were then
rinsed 3.times. with 1.times.PBS, including a 20 min rinse with 200
rpm shaking to remove unbound treatment molecule.
Platelet Preparation and Activation
Human whole blood was collected from healthy volunteers by
venipuncture following the approved Purdue IRB protocol and with
informed consent. The first 5 mL of blood was discarded as it can
be contaminated with collagen and other proteins, and approximately
15 mL was then collected into citrated glass vacutainers (BD
Bioscience). Blood was centrifuged in the glass tube for 20 min at
200.times.g at 20.degree. C. The top layer of the centrifuged
blood, the platelet rich plasma (PRP), was used for platelet
experiments. PRP (50 .mu.L/well) was added to the microplate and
allowed to incubate for 1 hr at room temperature without
shaking.
After 1 hour of incubation, the PRP was removed from each well and
added to a microcentrifuge tube containing 5 .mu.L ETP (107 mM
EDTA, 12 mM theophylline, and 2.8 .mu.M prostaglandin E1) to
inhibit further platelet activation. These tubes were spun at
4.degree. C. for 30 min at 1900.times.g to pellet the platelets.
The supernatant (platelet serum) was collected for ELISA studies to
test for the presence of platelet activation markers PF-4 and
Nap-2.
Platelet Adherence
After the PRP was removed from the wells of the collagen/treatment
coated plates, the wells were rinsed 3.times. with 0.9% NaCl for 5
min each shaking at 200 rpm. Platelet adherence was quantified
colormetrically or visualized fluorescently.
Colormetric Assay
140 .mu.L of a sodium citrate/citric acid buffer (0.1M, pH 5.4)
containing 0.1% Triton X-100 and 1 mg/mL p-nitrophenyl phosphate
was added to each well. The background absorbance was measured at
405 nm. The plate was then incubated for 40 min at room temperature
with shaking at 200 rpm. The Triton X-100 creates pores in the
cells, allowing p-nitrophenyl phosphate to interact with acid
phosphatase in the platelets to produce p-nitrophenol. After 40 min
of incubation, 100 .mu.L of 2M NaOH was added to each well. The pH
change stops the reaction by inactivating acid phosphatase, and
also transforms the p-nitrophenol to an optically active compound.
The absorbance was then read at 405 nm and correlated to the number
of adhered platelets. The results are shown in FIG. 41.
Fluorescent Assay
Adhered platelets were fixed by incubation with 4% paraformaldehyde
for 10 min at room temperature. The platelets were permeabilized
with 0.1% Triton X-100 for 5 min. Platelet actin was labeled by
incubation with phalloidin-AlexaFluor 488 (Invitrogen) containing
1% BSA for 30 min. The wells were rinsed 3.times. with 1.times.
PBS, and the adhered platelets were imaged using an upright
fluorescent microscope using a DAPI (4',6-diamidino-2-phenylindole)
filter.
See FIGS. 42 to 52 for results. Platelet aggregation on untreated
collagen surfaces is indicated by blurred images resulting from
clumped platelets. Without being bound by theory, it is believed
that clumping of platelets in the z-direction (perpendicular to the
plate surface) prevents image capture in one focal plane. On
treated surfaces, reduced platelet aggregation results in less
clumping (fewer platelets in the z-direction), and focused images
can be captured at the plate surface. These images show that
treatment with the synthetic peptidoglycans reduces adhesion of
platelet cells to collagen,
Detection of Platelet Activation Markers
The supernatant (platelet serum) obtained after pelleting the
platelets was used to determine released activation factors.
Platelet factor 4 (PF-4) and .beta.-thromboglobulin (Nap-2) are two
proteins contained within alpha granules of platelets which are
released upon platelet activation. Sandwich ELISAs were utilized in
order to detect each protein. The components for both sandwich
ELISAs were purchased from (R&D Systems) and the provided
protocols were followed. The platelet serum samples were diluted
1:10,000-1:40,000 in 1% BSA in 1.times.PBS so the values fell
within a linear range. The results shown in FIGS. 39 and 40 show
that treatment with synthetic peptidoglycans decreases platelet
activation by collagen I.
EXAMPLE 37
Inhibition of Platelet Binding and Platelet Activation to Collagen
Type III and Type I
The method according to EXAMPLE 36 was used with the following
modification.
Microplate Preparation
Type I collagen (rat tail collagen, BD Biosciences) and type III
collagen (Millipore) were combined on ice with NaOH, 1.times.PBS,
and 10.times.PBS to physiological conditions. The total collagen
concentration was 1 mg/mL with 70% type I collagen and 30% type III
collagen. 30 .mu.L of the collagen solution was pipetted into each
well of a 96-well plate. The plate was incubated at 37.degree. C.
in a humidified incubator for one hour, allowing a gel composed of
fibrillar collagen to form in the wells. The wells were rinsed
3.times. with 1.times.PBS.
Peptidoglycan was diluted in 1.times.PBS at concentrations of 25
.mu.M and 50 .mu.L solution was added to the collagen coated wells.
Controls of GAG, peptide, or PBS were also added to collagen coated
wells as controls. Combinations of peptidoglycan or peptide were
composed of 25 .mu.M of each molecule in 1.times.PBS. Treatments
were incubated at 37.degree. C. with shaking at 200 rpm for 30 min.
Wells were then rinsed 3.times. with 1.times.PBS, including a 10
min rinse with 200 rpm shaking to remove unbound treatment
molecule.
The results of the platelet activation inhibition measurements
shown in FIG. 54 demonstrate that the synthetic peptidoglycans
inhibit platelet cell activation by a mixture of collagen Type I
and Type III.
The results shown in FIG. 55 demonstrate that the peptidoglycans
inhibit platelet cell binding to collagen Type 1 and Type III
mixtures.
SEQUENCE LISTINGS
1
18120PRTArtificial SequenceSynthetic peptide 1Arg Arg Ala Asn Ala
Ala Leu Lys Ala Gly Glu Leu Tyr Lys Ser Ile 1 5 10 15 Leu Tyr Gly
Cys 20 211PRTArtificial SequenceSynthetic peptide 2Arg Leu Asp Gly
Asn Glu Ile Lys Arg Gly Cys 1 5 10 315PRTArtificial
SequenceSynthetic peptide 3Ala His Glu Glu Ile Ser Thr Thr Asn Glu
Gly Val Met Gly Cys 1 5 10 15 430PRTArtificial SequenceSynthetic
peptide 4Asn Gly Val Phe Lys Tyr Arg Pro Arg Tyr Phe Leu Tyr Lys
His Ala 1 5 10 15 Tyr Phe Tyr Pro Pro Leu Lys Arg Phe Pro Val Gln
Gly Cys 20 25 30 510PRTArtificial SequenceSynthetic peptide 5Cys
Gln Asp Ser Glu Thr Arg Thr Phe Tyr 1 5 10 69PRTArtificial
SequenceSynthetic peptide 6Thr Lys Lys Thr Leu Arg Thr Gly Cys 1 5
730PRTArtificial SequenceSynthetic peptide 7Gly Leu Arg Ser Lys Ser
Lys Lys Phe Arg Arg Pro Asp Ile Gln Tyr 1 5 10 15 Pro Asp Ala Thr
Asp Glu Asp Ile Thr Ser His Met Gly Cys 20 25 30 89PRTArtificial
SequenceSynthetic peptide 8Ser Gln Asn Pro Val Gln Pro Gly Cys 1 5
913PRTArtificial SequenceSynthetic peptide 9Ser Tyr Ile Arg Ile Ala
Asp Thr Asn Ile Thr Gly Cys 1 5 10 1011PRTArtificial
SequenceSynthetic peptide 10Ser Tyr Ile Arg Ile Ala Asp Thr Asn Ile
Thr 1 5 10 118PRTArtificial SequenceSynthetic peptide 11Lys Glu Leu
Asn Leu Val Tyr Thr 1 5 1210PRTArtificial SequenceSynthetic peptide
12Lys Glu Leu Asn Leu Val Tyr Thr Gly Cys 1 5 10 1312PRTArtificial
SequenceSynthetic peptide 13Gly Ser Ile Thr Thr Ile Asp Val Pro Trp
Asn Val 1 5 10 1414PRTArtificial SequenceSynthetic peptide 14Gly
Ser Ile Thr Thr Ile Asp Val Pro Trp Asn Val Gly Cys 1 5 10
1521PRTArtificial SequenceSynthetic peptide 15Gly Arg Arg Ala Asn
Ala Ala Leu Lys Ala Gly Glu Leu Tyr Lys Ser 1 5 10 15 Ile Leu Tyr
Gly Cys 20 1612PRTArtificial SequenceSynthetic peptide 16Gly Ser
Tyr Ile Arg Ile Ala Asp Thr Asn Ile Thr 1 5 10 179PRTArtificial
SequenceSynthetic peptide 17Gly Glu Leu Tyr Lys Ser Ile Leu Tyr 1 5
1820PRTArtificial SequenceSynthetic peptide 18Arg Arg Ala Asn Ala
Ala Leu Lys Ala Gly Glu Leu Tyr Lys Ser Ile 1 5 10 15 Leu Tyr Gly
Ser 20
* * * * *
References